A reliable and reproducible model in vitro for swine enteric coronaviruses infection would be intestinal models that support virus replication and can be long-term cultured and manipulated experimentally. Here, we designed a robust long-term culture system for porcine intestinal organoids from the intestinal crypt or single LGR5⁺ stem cell by combining previously defined insights into the growth requirements of the intestinal epithelium of humans. We showed that long-term cultured swine intestinal organoids were expanded in vitro for more than 6 months and maintained the potential to differentiate into different types of cells. These organoids were successfully infected with porcine enteric coronavirus, including porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), and were capable of supporting virus replication and progeny release. RNA-seq analysis showed robust induction of transcripts associated with antiviral signaling in response to enteric coronavirus infection, including hundreds of interferon-stimulated genes and cytokines. Moreover, gene set enrichment analysis indicated that PEDV infection could suppress the immune response in organoids. This 3D intestinal organoid model offers a long-term, renewable resource for investigating porcine intestinal infections with various pathogens.

The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus’s impact on the swine industry. IMPORTANCE: Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1β, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry worldwide. Our previous study had indicated that proprotein convertase subtilisin/kexin type 9 (PCSK9) was a responsive gene in porcine alveolar macrophages (PAMs) upon PRRSV infection. However, whether PCSK9 impacts the PRRSV replication and how the PRRSV modulates host PCSK9 remains elusive. Here, we demonstrated that PCSK9 protein suppressed the replication of both type-1 and type-2 PRRSV species. More specifically, the C-terminal domain of PCSK9 was responsible for the antiviral activity. Besides, we showed that PCSK9 inhibited PRRSV replication by targeting the virus receptor CD163 for degradation through the lysosome. In turn, PRRSV could down-regulate the expression of PCSK9 in both PAMs and MARC-145 cells. By screening the nonstructural proteins (nsps) of PRRSV, we showed that nsp11 could antagonize PCSK9’s antiviral activity. Furthermore, mutagenic analyses of PRRSV nsp11 revealed that the endoribonuclease activity of nsp11 was critical for antagonizing the antiviral effect of PCSK9. Collectively, our data provide further insights into the interaction between PRRSV and the cell host and offer a new potential target for the antiviral therapy of PRRSV.