Utility Evaluation Of Porcine Enteroids As Porcine Enterocoronavirus Infection Model In Vitro

Intestinal enteroids,which partially recreate the structure and function of intestinal villi-crypts,have many physiological similarities to the intestinal tissues in vivo.Enteroids have advantages in studying the interactions between intestines and enteric pathogens.Porcine Epidemic Diarrhea Virus(PEDV)and Porcine Deltacoronavirus(PDCoV)are novel emerging enteric coronavirus of pigs,and cause severe diarrhea in piglets,which hinder the swine industry.This study developed a robust PDCoV infection system in vitro,and further investigated the pathophysiological changes after infection and differences with the traditional model IPEC-J2 cells.The results provided a theoretical foundation for further research on the pathogenic mechanism and treatment of porcine enterocoronavirus in the future.1.Establishment of 3D and 2D porcine enteroids culture systemsThis study established a relatively simple method of crypt isolation and in vitro culture,and the cultured enteroids had almost 100%viability.The proportion of EdU-positive cells and clone-forming efficiency of enteroids derived from duodenum are significantly higher than those of the other two intestinal segments.Duodenum enteroids can survive for 4 months with no anomalies in their growth rate,morphology and pathogen infection rates during culture.The two-dimensional enteroids we cultured could form a tight confluent monolayer in three days and allow the virus to easily enter from the apical surface of the mucosa.Similar to three-dimensional enteroids,it contains five types intestinal epithelium cell type:absorption cells,enteroendocrine cells,goblet cells,Paneth cells and stem cells.During the differentiation process,the proportion of CD44-positive stem cell and progenitor cell populations decreased by 30.7%and the transcription level of stem cell marker gene Lgr5 was significantly reduced,while the population of absorbed and secreted cells increased accordingly.In short,two-dimensional organoids can be used as a system to simulate the gut.2.Evaluation of porcine enteroids as a infection model of PEDV and PDCoV in vitroBoth PEDV and PDCoV can infect two-dimensional porcine enteroids,and induced+4 stem cell marker gene Bmil and goblet cell marker gene TFF3 to be significantly up-regulated after 24 hours of infection,suggesting that the two viruses could regulate proliferation and differentiation of enteroids in the same way.There is no significant difference in the replication level of PDCoV in enteroids of passages 2,8 and 14,and Sox9 and Villinl positive intestinal epithelial cells may be the target cells of PDCoV.The transcriptional changes of mucosal immunity and antiviral genes of enteroids infected with PDCoV were significantly higher than those of IPEC-J2 cells.We conclude that the 2-D enteroids derived from porcine jejunum can be used as an in vitro multicellular model for investigation of pathogenesis and host immune responses to porcine enteric pathogens,such as PDCoV.