| Porcine deltacoronavirus（PDCoV）,a recently discovered porcine enteropathogenic coronavirus,mainly causes acute diarrhea in newborn piglets,which has posed a serious threat to the global pork industry.The main site of PDCoV infection in vivo is porcine small intestine,which can infect cells from broad species including human,porcine and chicken in vitro,and has a potential capacity of interspecies transmission.Therefore,it is particularly important to clarify the molecular mechanism that determines the cell and intestinal tissue tropism of PDCoV.However,the lack of an in vitro model that can mimic the complex structure and function of intestinal epithelium in vivo significantly limits the study of the interactions between PDCoV infection and host intestinal epithelium.Recently,an enteroid model established in vitro that can recapitulate the highly complex physiological properties of intestinal epithelium in vivo,provides an ideal in vitro model for elucidating the pathogenic mechanism of PDCoV.In this study,to evaluate whether PDCoV can infect porcine intestinal enteroids,porcine intestinal enteroids derived form cypt stem cells were isolated and cultured in vitro.PDCoV infection in porcine enteroids was analysed using RT-qPCR,TCID50 and IFA,respectively.The results showed that porcine intestinal enteroids were susceptible to PDCoV infection and were capable of supporting the release of progeny virus particles(viral titers reached up to 105.33 TCID50/mL).To investigate the target cell types of PDCoV infection in the small intestine epithelium,the results of IFA revealed that PDCoV could infect multiple functional intestinal epithelia such as enterocytes,stem cells and goblet cells.Furthermore,to explore whether porcine intestinal enteroids can mimic the intestinal tissue tropism of PDCoV infection in vivo,the replication of PDCoV in jejunal enteroids was significantly higher than that of duodenal enteroids and colonoids detected by RT-qPCR,TCID50 and IFA,which was consistent with the results of PDCoV infection in vivo.In order to further explore the molecular mechanism of PDCoV intestinal tissue tropism,the analysis of RNA sequencing（RNA-Seq）and RT-qPCR demonstrated that the transcriptional profiles of enteroids derived from different intestinal segments exhibited a segment-specific expression pattern,and the differential expression of cellular receptor porcine aminopeptidase N（pAPN）in different intestinal segments was closely related to the intestinal tissue tropism of PDCoV,while the expression levels of host proteases known to be associated with coronavirus infection and interferon（IFN）receptors were not correlated to the intestinal tissue tropism of PDCoV.To further clarify whether the IFN immune responses are related to the intestinal tissue tropism of PDCoV,we found that the expression levels of antiviral genes in jejunal enteroids induced by PDCoV were significantly higher than those of duodenal enteroids and colonoids,which was consistent with the level of PDCoV replication in enteroids,suggesting that the IFN immune responses are not related to the intestinal tissue tropism of PDCoV.In addition,we monitored the expression of IFN in enteroids after PDCoV infection,the results indicated that typeⅠand typeⅢIFNs were markedly induced at the late stage of PDCoV infection,and the exogenous IFN-λ1 treatment significantly inhibited PDCoV replication in enteroids,which further excluded that the uncorrelation between the IFN responses and the intestinal tissue tropism of PDCoV was because of the tolerance of PDCoV to IFN.In summary,we revealed the molecular mechanism of PDCoV intestinal tissue tropism,which provides new insights for elucidating the pathogenesis of PDCoV.Moreover,our results indicated that porcine intestinal enteroids can be used as a good in vitro model to study the pathogenesis of porcine enteric coronavirus,which lays a foundation for an-depth exploration of the molecular biology of enteric viruses. |
