Utility Evaluation of Porcine Enteroids as PDCoV Infection Model in vitro.

Hao Luo,Yunlu Chen,Xiaoliang Li,Jidong Xu,Zhenning Zhang,Tingjun Wang,Jingyou Zheng,Weihuan Fang,Ying Shan,Min Yue

doi:10.3389/fmicb.2020.00821

Abstract

Porcine deltacoronavirus (PDCoV) is a novel emerging enteric coronavirus found in pigs. Intestinal enteroids, which partially recreate the structure and function of intestinal villi-crypts, have many physiological similarities to the intestinal tissues in vivo. Enteroids exhibit advantages in studying the interactions between intestines and enteric pathogens. To create a novel infection model for PDCoV, we developed an in vitro system to generate porcine intestinal enteroids from crypts of duodenum, jejunum, and ileum of pigs. Enterocytes, enteroendocrine cells, Paneth cells, stem cells, proliferating cells, and goblet cells were found in the differentiated enteroids. Replication of PDCoV was detected in the cultured enteroids by immunofluorescence and quantitative RT-PCR. Double immunofluorescence labeling demonstrated that PDCoV was present in Sox9-positive intestinal cells and Villin1-positive enterocytes. There were multiple cellular responses shown as changes of transcription of genes related to mucosal immunity, antiviral genes, and marker genes of stem cells and other cells in the enteroids infected with PDCoV. We conclude that the 2-D enteroids derived from porcine jejunum can be used as an in vitro multicellular model for the investigation of pathogenesis and host immune responses to porcine enteric pathogens, such as PDCoV.

Highlights

Porcine enteroviruses are a group of highly contagious pathogens that cause serious economic losses worldwide
The colony-forming efficiency (CFE) of the enteroids derived from duodenum was significantly higher than those from jejunum and ileum (p < 0.001) (Figure 2D)
The jejunal enteroids were passaged more than 14 times (P14) and there were no morphological changes even after 14 passages, as compared with earlier generations (Figure 2E)

Summary

Introduction

Porcine enteroviruses are a group of highly contagious pathogens that cause serious economic losses worldwide. The porcine intestinal epithelial cell lines SD-PJEC (Sun et al, 2012) and IPEC-J2 (Jung et al, 2018) are most commonly used for in vitro studies and characterization of the interaction between intestinal cells and enteroviruses These cell lines consist of only a single type of cells, lacking the complexity and physiology of multiple cells in the native intestinal tracts. These cell lines are prone to spontaneous mutation upon repeated passages, reducing their applicability (Yin et al, 2019). More representative models comprising multiple cells mimicking the intestinal tracts are required for in vitro studies

Methods

Results

Conclusion