Long-Term Expansion of Porcine Intestinal Organoids Serves as an in vitro Model for Swine Enteric Coronavirus Infection

Min Zhang,Changlong Liu,Lilei Lv,Liwei Li,Guangzhi Tong,Guoxin Li,Wu Tong,Fei Gao,Yanhua Li,Lingxue Yu,Hongming Cai,Yifeng Jiang

doi:10.3389/fmicb.2022.865336

Abstract

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.

Highlights

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV) are major pathogenic swine enteric viruses, which belong to the family Coronaviridae
The crypts of duodenum, jejunum, and ileum from 10-day old pig were isolated and embedded in basement membrane extract (Matrigel) and covered with porcine intestinal organoids (PIO) medium, which is modified from human intestinal organoids culture system
The results showed that the numbers of porcine epidemic diarrhea virus (PEDV) genomes inner cells of organoids were substantially increased after PEDV infection (Figure 3A), indicating that PEDV can infect long-term culture porcine intestinal organoids

Summary

Introduction

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV) are major pathogenic swine enteric viruses, which belong to the family Coronaviridae. PEDV, TGEV, and PDCoV are highly contagious and are a major cause of illness and death in piglets (Jung and Saif, 2015; Jung et al, 2016). These viruses can cause endemics or large-scale epidemics in major pig-producing countries, which lead to severe economic losses in the swine industry. Several swine intestine epithelial cell lines were developed like IPEC-J2 and IPI-21, which derived from pig jejunum and ileum, respectively. The viral infectivity in IPEC-J2 and IPI21 cells are controversial, and it is increasingly evident that these cell models are not sufficient to fully understand swine enteric CoVs diseases (Zhao et al, 2014; Wang et al, 2019).

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Conclusion