Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response

Li, Liang; Fu, Fang; Guo, Shan-Shan; Wang, Hongfeng; He, Xijun; Xue, Mei; Yin, Liang; Feng, Li; Liu, Pinghuang

doi:10.1128/jvi.01682-18

Cited by 66 publications

(109 citation statements)

References 42 publications

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“…To determine whether PEDV elicits an endogenous IFN-λ response in Vero E6 cells following infection, we initially infected Vero E6 cells with PEDV at MOI = 0.1 and monitored the IFN-λ expression. Compared with a mock uninfected control, PEDV did not increase the expression of IFNλ transcripts as observed until 12 hpi, and then gradually induced IFN-λ expression, indicating that PEDV infection elicits type III IFN expression at the late stage of infection instead of the early stage of infection in the Vero E6 cells (Figure 1A), which was consistent with the results in porcine enteroids (Li et al, 2019). And PEDV propagated efficiently in Vero E6 cells by quantifying viral genomes and titers (Figures 1B,C).…”

Section: Pedv Replicated Well Despite the Induction Of Endogenous Ifnsupporting

confidence: 88%

“…Among three types of IFNs (types I, II, and III), type III IFN-lambda (IFN-λ) primarily acts on mucosal surfaces, including epithelial surfaces of the liver, respiratory, and gastrointestinal systems, and plays vital roles in controlling viral infection within mucosal surfaces (Mordstein et al, 2010;Pott et al, 2011;Lazear et al, 2015). We and other groups previously demonstrated that porcine IFN-λdisplays powerful antiviral activity against PEDV infection in both Vero E6 cells and porcine intestinal epithelia (Li et al, 2017(Li et al, , 2019. PEDV has evolved multiple strategies to escape IFN responses, including the degradation of STAT1 and the suppression of type I IFN production (Guo et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

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The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis

Shan

Xue

et al. 2020

Front. Microbiol.

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Porcine epidemic diarrhea virus (PEDV) is an economically important pathogen that has evolved several mechanisms to evade type I IFN responses. Type III interferon (IFN-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit PEDV infection in vitro. However, how PEDV escapes IFN-λ antiviral response remains unclear. In this study, we found that PEDV infection induced significant IFN-λ expression in type I IFN-defective Vero E6 cells, but virus-induced endogenous IFN-λ did not reduce PEDV titers. Moreover, we demonstrated that PEDV escaped IFN-λ responses by substantially upregulating the suppressor of cytokine signaling protein 1 (SOCS1) expression, which impaired the induction of IFN-stimulated genes (ISGs) and dampened the IFN-λ antiviral response and facilitated PEDV replication in Vero E6 cells. We further showed that PEDV infection increased SOCS1 expression by decreasing host miR-30c-5p expression. MiR-30c-5p suppressed SOCS1 expression through targeting the 3 ′ untranslated region (UTR) of SOCS1. The inhibition of IFN-λ elicited ISGs expression by SOCS1 was specifically rescued by overexpression of miR-30c-5p. Collectively, our findings identify a new strategy by PEDV to escape IFN-λ-mediated antiviral immune responses by engaging the SOCS1/miR-30c axis, thus improving our understanding of its pathogenesis.

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Section: Pedv Replicated Well Despite the Induction Of Endogenous Ifnsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis

Shan

Xue

et al. 2020

Front. Microbiol.

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“…The results of dynamic invasion showed that the virus invaded Vero cells 100% within 60 min, while the invasion efficiency of the IPEC-J2 cells was only approximately 30%. Although IPEC-J2 cells are considered the host cells of PEDV, the cell lines cultured in vitro lost their polar growth state in vivo [66], which possibly affects the viral recognition and reduces the infection efficiency of the virus. GDS01 showed a lower invasion rate than GDS09, but there was no significant difference.…”

Section: Figure 8 Pedv Traffics To Lysosome Via Endosomes a Bmentioning

confidence: 99%

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

Wang

She

et al. 2020

Vet Res

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With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin-and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome-late endosome-lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article'

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“…We conclude that porcine jejunum organoids more closely resemble jejunum tissue than IPECJ2 and provide a robust model for gene expression studies for at least 12 weeks of culture. As such they provide an advanced model for mechanistic studies on host-microbe interactions and intestinal physiology [41]. Organoids are also likely to avoid changes in glycosylation patterns seen in cancer or transformed cell models.…”

Section: Discussionmentioning

confidence: 99%

Congruence of location-specific transcriptional programs in intestinal organoids during long-term culture

Hee

Madsen²,

Smidt

et al. 2019

Preprint

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The emergence of intestinal organoids, as a stem cell-based self-renewable model system, has led to many studies on intestinal development and cell-cell signaling. However, potential issues regarding the phenotypic stability and reproducibility of the methodology during culture still needs to be addressed for different organoids. Here we investigated the transcriptomes of intestinal organoids derived from the same pig as well as batch-to-batch variation of organoids derived from different pigs over long-term passage. The set of genes expressed in organoids closely resembled that of the tissue of origin, including location specific functions, for at least 17 passages. Minor differences in gene expression were observed between individual organoid cultures. In contrast, most tissue-specific genes were not expressed in the transformed jejunum cell line IPECJ2, which also showed gene expression consistent with cancer phenotypes. We conclude that intestinal organoids provide a robust and stable model for translational research with clear advantages over transformed cells. Methods Intestinal organoid generationJejunum tissue segments were obtained from control piglets used for another study, following guidelines of the animal ethics committee of Wageningen University. Two 5-week-old piglets were used for generating organoids following procedures previously described [11,13]. Briefly, a 2 cm section of the mid-jejunum was dissected and placed in ice-cold PBS. After opening the sections longitudinally, jejunum segments were washed three times in ice-cold PBS and villi removed by carefully scraping with a scalpel. Small sections of the mucosa were cut from the muscle layer, divided into small cubes, and transferred into ice-cold PBS containing 30 mM EDTA and incubated with rotation at room temperature for 15 min. The PBS -EDTA was then replaced and incubation continued for 10 min at 37 °C. After washing in ice-cold DMEM supplemented with 5% penicillin/streptomycin (Gibco, Thermo Fisher Scientific), the crypts were dissociated by rigorous vortexing and passed through a 100 µm strainer into cold DMEM containing 5% foetal bovine serum (FBS, v/v). Crypts were pelleted by centrifugation at 300 x g for 5 min, and suspended in Matrigel (Basement Membrane, Growth factor reduced, REF 356231, Corning, Bedford, MA, USA). To improve surface tension, empty 24-well plates were pre-incubated overnight at 37 °C. Matrigel containing crypts was then plated at 7 domes per well (approx. 35 µl per well) and inverted to polymerize at 37 °C. After polymerization, 600 µl F12 cell culture medium (Gibco) was added, supplemented with 100 μg/ml primocin (Invivogen), 10 mM HEPES (HyClone), 1 × B-27 (Gibco), 1.25 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D systems), 15 nM gastrin, 10 mM nicotinamide, 10 μM p38 MAPK inhibitor (Sigma), 600 nM TGFβ receptor inhibitor A83-01, and conditioned media for recombinant Noggin (15% v/v), Spondin (15% v/v), and Wnt3A (30% v/v) provided by dr. Kuo and Hubrecht Institute (Utrecht, the Ne...

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Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response

Cited by 66 publications

References 42 publications

The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis

The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

Congruence of location-specific transcriptional programs in intestinal organoids during long-term culture

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