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Identification of cell types and transcriptome landscapes of PEDV infected porcine small intestine using single-cell RNA-sequencing

ABSTRACT: Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. Here, single-cell RNA-sequencing was used to perform a systematic analysis of pig small intestines infected with PEDV for the first time. Multiple cell types were identified by representative markers, including the unique marker DNAH11 of tuft cells. Meanwhile, the goblet and tuft cells were also susceptible to PEDV except enterocytes. PEDV infection obviously upregulated REG3G, which significantly inhibited virus replication. Notably, IFN-DELTAs in goblet and enterocyte progenitor cells were increased in virus infected piglet, and IFN-DELTA5 could induce GBP1, ISG15, OAS2 and IFITM1 dramatically raised in IPEC-J2 cells and restricted PEDV replication. Complement molecules were mainly expressed in intestinal cells excepting tuft cells, but PEDV decreased C3, C4A, and C5 in enterocytes, thus escaping the antiviral effect of C3. Finally, enterocytes expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in porcine enterocyte cells. In summary, this study systematically studied the response of different cell types in small intestine of pigs after PEDV infection, which deepened the understanding of viral pathogenesis.

ORGANISM(S): Sus scrofa

PROVIDER: GSE175411 | GEO | 2022/04/27

REPOSITORIES: GEO

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A recombination-resistant genome for live attenuated and stable PEDV vaccines by engineering the transcriptional regulatory sequences

Project description:Porcine epidemic diarrhea virus (PEDV) is a deadly coronavirus for neonatal piglets and no effective vaccines are available. Transcriptional regulatory sequences (TRSs) are critical in regulating coronavirus discontinuous transcription. Also, TRSs contribute to a high recombination rate of coronaviruses, leading to difficulty in developing safe live vaccines. We hypothesize that recoding the TRS core sequences (TRS-CS) of PEDV can make the recombination impossible between the engineered vaccine virus and field strains or wildtype viruses. We used an infectious clone-derived reporter PEDV, dORF3-EGFP, as the backbone to generate a remodeled TRS (RMT) mutant that carries the recoded leader and body TRS-CSs. The RMT and dORF3-EGFP showed comparable replication efficiency in Vero cells. However, the incompatibility between the rewired and wildtype TRS-CSs led to few EGFP in RMT-infected cells. Furthermore, RMT and dORF3-EGFP had a similar attenuated phenotype, replication efficiency, and protective immunogenicity in neonatal pigs. RNA sequencing analysis indicated that EGFP transcription directed by the heterogenous TRS-CSs was significantly reduced to an extremely low level. Meanwhile, recombinant viruses were not detected in Vero cells and in pigs that were co-infected with RMT and a PEDV S-INDEL strain, Iowa106. In vitro and in vivo passaging of the RMT did not result in reversion mutations in the rewired TRS-CSs, introduced gaps, and disrupted wildtype TRSs. In summary, the RMT mutant was resistant to recombination and genetically stable and can be further optimized (e.g., deletion of the EGFP) to serve as a platform to develop safe PEDV live attenuated vaccines.

2023-11-04 | GSE246991 | GEO

Coronavirus replication is regulated by both host and viral RNA N6–methyladenosine modifications

Project description:N6–methyladenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, and it plays an important role in RNA metabolism and function. Recent studies have revealed that viral RNA m6A modification can play an anti-viral or pro-viral role depending on the virus. However, whether m6A methylation can modulate replication of coronaviruses, which cause some severe human and animal diseases such as Coronavirus Disease 2019 (COVID-19), remains unknown. Here, we determined that m6A modifications of porcine epidemic diarrhea coronavirus (PEDV) are exclusively located in the N gene at the 3’-end of the genome, and that PEDV infection alters the expression pattern of some host proteins involved in m6A modification. Depletion of m6A demethylases significantly increased PEDV replication and gene expression whereas knockdown of m6A methyltransferases slightly decreased PEDV infection. Interestingly, m6A binding proteins YTHDF 2 and 3 significantly inhibited PEDV replication whereas YTHDF1 has the opposite effect. When the major m6A sites in the N gene were mutated, the resultant recombinant PEDVs and PEDV replicons had a significant increase in replication and gene expression. This study illustrates that (i) addition of m6A to PEDV RNA inhibits viral replication, and (ii) both host and viral m6A machinery regulate coronavirus replication.

2020-05-22 | GSE151020 | GEO

Long-term Expansion of Porcine Intestinal Organoids serves as an Ex Vivo Model for Swine Enteric Coronavirus infection

Project description:We designed a long-term culture system for porcine intestinal organoids from intestinal crypt or single Lgr5+ stem cells by combining previously defined insights in the growth requirements of intestinal epithelium of human and mouse. We showed that long-term cultured swine intestinal organoids were expanded in vitro more than six months at least 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). RNA-seq analysis showed that robust induction of transcripts associated with antiviral signaling in response to enteric coronavrius infection, including a number of interferon-stimulated genes and cytokines. Moreover, gene set enrichment analysis indicated that PEDV infection could suppress immune response in organoids. This 3D intestinal organoid model offers a long-term, renewable resource for investigating porcine intestinal infections with a variety of pathogens.

2021-08-19 | GSE182240 | GEO

Coronavirus replication is controlled by both host and viral RNA m6A methylations

Project description:N6–methyladenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, and it plays an important role in RNA metabolism and function. Recent studies have revealed that viral RNA m6A modification can play an anti-viral or pro-viral role in virus life cycle. However, whether m6A methylation can modulate replication of coronaviruses, which contain the largest known RNA genomes, remains elusive. Here, we determined that m6A modifications of porcine epidemic diarrhea coronavirus (PEDV) are exclusively located in the N gene at the 3’-end of genome, and that PEDV infection alters the expression pattern of host proteins involved in m6A modification. Depletion of m6A demethylases significantly increased PEDV replication and gene expression whereas knockdown of m6A methyltransferases slightly decreased PEDV infection. Interestingly, m6A binding proteins YTHDF 2 and 3 significantly inhibited PEDV replication whereas YTHDF1 has an opposite effect. When the major m6A sites in the N gene were mutated, the resultant recombinant PEDVs and PEDV replicons had a significant increase in replication and gene expression. This study illustrates that (i) addition of m6A to PEDV RNA inhibits viral replication, and (ii) both host and viral m6A machinery regulates coronavirus replication.

2022-12-01 | GSE141291 | GEO

comparative proteome analysis of serum exosomes from newborn piglets inresponse to porcine epidemic diarrhea virus infection

Project description:The immune system is thought to be fragile in the neonate, which is susceptible to pathogens. Exosomes are a type of vehicles existing in the body fluid and participate in many biological processes, especially the immune response. Inorder to investigate the roles that exosomes may play during virus infection in the neonate, porcine epidemic diarrhea virus (PEDV), a devastating enteric virus to newborn piglets, was selected for infection. Serum exosomes were then isolated from the newborn-piglets infected or mock-infected with PEDV and followed by a label-free LC-MS/MS based comparative quantitative proteomic analysis. Among 441 proteins detected in the serum exosomes, there were lots of complement proteins. The expression level of the complement C3, C6 and CFB suffered drastic changes due to PEDV infection. After the confirmation by western-blot assay, we then investigated the function of these exosomes on PEDV infection and discovered that the exosomes from mock-infected newborn piglets restricted PEDV infection but this inhibition disappeared after exosomes were heat-inactivated, suggesting that the complement is one of the key antiviral molecules. These findings will facilitate the understanding of the antiviral response of the neonate mediated by exosomes

2019-04-30 | PXD010762 | Pride

Identification of host proteins interacting with the E protein of porcine epidemic diarrhea virus

Project description:Porcine epidemic diarrhea (PED) is an acute, highly contagious, and high-mortality enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV). PEDV is globally endemic and causes substantial economic losses in the swine industry. The PEDV E protein is the smallest structural protein with high expression levels that interacts with the M protein and participates in virus assembly. However, how the host proteins interact with E proteins in PEDV replication remains unknown. We identified host proteins that interact with the PEDV E protein using a combination of PEDV E protein-labeled antibody co-immunoprecipitation and tandem liquid-chromatography mass-spectroscopy (LC-MS/MS).

2024-03-05 | PXD050352 | Pride

Compare of differential expression profiles of PEDV-susceptible and non-susceptible cell lines at the transcriptome level to identify key molecules associated with PEDV entry

Project description:Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that poses a serious threat to the global pig industry. Despite extensive research efforts, the functional receptor for PEDV remains unclear. In this study, we identified susceptible and non-susceptible cell lines to PEDV infection, and performed RNA-seq analysis on these cell lines. Using Weighted Gene Co-expression Network Analysis (WGCNA), we have identified the key pathways that correlated with the PEDV entry pathway. We found that cholesterol, sterols, and lipid transport and homeostasis were strongly correlated with PEDV entry, suggesting a potential role for cholesterol in PEDV entry. We then treated susceptible cell lines with a cholesterol transport inhibitor and found that inhibition of cholesterol transport could significantly inhibit PEDV entry in these cells. Together, our results suggest that cholesterol transport may play a critical role in the entry of PEDV into susceptible cells, and that targeting cholesterol transport may represent a potential strategy for controlling PEDV transmission. Our findings provide new insights into the mechanism of PEDV entry and may pave the way for the development of new therapeutic strategies against this economically important virus. Further studies are warranted to elucidate the detailed mechanism of PEDV entry, and to explore the potential of cholesterol transport inhibitors as a means of controlling PEDV transmission.

2023-07-06 | GSE236338 | GEO

Transcriptomes of adult sorted Epcam+ CD44- enterocytes from small intestine of wildtype and CmafLEC-KO mice

Project description:Small intestinal villi are highly specialized structural and functional units uniquely adapted to the absorption of nutrients in higher vertebrates. Intestinal villus enterocytes are organized in spatially resolved “zones” dedicated to specialized tasks, such as anti-bacterial protection, and absorption of amino-acids, carbohydrates and lipids. However, the molecular mechanisms for the establishment and maintenance of villus intestinal epithelial zonation are incompletely understood. Here, we show that transcription factor C-Maf is highly expressed in mature lower and mid-villus small intestinal enterocytes, where it is induced in response to BMP signaling. In vivo depletion of C-Maf in the adult enterocytes perturbed the villus epithelial zonation transcriptional program and enhanced the expression of genes involved in carbohydrate metabolism and bile acid regulation and transport, while suppressing genes related to amino acid transport and lipid metabolism. Loss of C-Maf under homeostatic conditions had no effect on body weight due to compensatory small intestinal remodeling, accompanied by increased generation of tuft cells and goblet cell hyperplasia. However, upon challenge with anti-metabolite therapy, C-Maf inactivation impaired body weight recovery, resulting in reduced survival, due to delayed enterocyte maturation. Our results identify a novel role for C-Maf in maintaining the zonation program of differentiated intestinal epithelium, by balancing absorptive versus secretory cell differentiation in the small intestine, while highlighting the importance of coordination between stem/progenitor cell and enterocyte differentiation programs for intestinal regeneration.

2022-08-18 | GSE189852 | GEO

Epithelial cell diversity in small intestine of pigs

Project description:To establish better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.

2023-01-18 | GSE208613 | GEO

Single-cell RNA sequencing and total RNA sequencing data, respectively, evaluating the expression of selective genes from C57BL/6 mouse-derived small intestinal organoids and whole ileal tissue from naïve Il22Ra1fl/fl;Defa6-cre+/- mice

Project description:All intestinal epithelial lineages express the IL-22 receptor; however, it is not known whether IL-22-dependent regulation of small intestinal host defense is dependent on ISCs or a specific epithelial lineages. To examine differences in gene expression among small intestinal enterocytes, single cell RNA sequencing was performed on primary small intestinal organoids derived from naïve C57BL/6 mice. Our scRNA-seq results demonstrate that Paneth cells express the highest level of Il22ra1 when compared to ISCs as well as enterocyte, goblet, tuft, and endocrine lineages. Additionally, total RNA sequencing (RNA-seq) of the terminal ileum of naïve Il22Ra1fl/fl;Defa6-cre+ (Paneth cell-specific IL22Ra1 knockout) and littermate cre- mice was performed. Our sequencing data revealed reduced expression of Paneth cell-specific Lyz1, Mmp7, and select alpha-defensin antimicrobial peptides but increased expression of IL-22 inducible Reg3g and serum amyloid genes (Saa1 and Saa3) in the terminal ileum of naïve Il22Ra1fl/fl;Defa6-cre+ mice

2020-10-14 | GSE159423 | GEO

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