哺乳动物重组病毒3型（MRV3）是引起包括猪在内的多种哺乳动物患病的胃肠炎病毒，已从严重腹泻的仔猪中分离得到。然而，猪源性细胞在口服MRV3感染模型中的应用很少被研究。本研究旨在建立猪肠脏器培养物（PIOs）并检测它们在体外作为MRV3肠道感染模型的敏感性。PIOs由迷你猪空肠部位分离并建立。利用聚合酶链式反应（PCR）和免疫荧光分析（IFAs）对建立的PIOs进行表征，以确认其表达小肠特异基因和蛋白质，如Lgr5、LYZI、Mucin-2、ChgA和Villin。通过将PIOs分散在培养板上以展示其顶层表面，获得单层PIOs和三维（3D）PIOs，并将其接种MRV3 2小时后，用Dulbecco磷酸盐缓冲盐水清洗，并进行观察。通过PCR和IFA确认病毒感染情况。我们进行定量实时逆转录聚合酶链式反应，评估与肠道上皮基因和抗病毒活性相关的病毒拷贝数和基因表达的变化。建立的PIOs具有肠道培养物的分子特征。感染的PIOs表现出延迟增殖和结构破坏的现象。此外，MRV3感染改变了与肠道上皮细胞和抗病毒活性相关的基因表达，这些影响在2D和3D模型中均有观察到。此外，两种模型的上清液中，病毒拷贝数随时间的推移而增加。我们认为，PIOs可以成为研究MRV3感染机制的体外模型，有助于药物的开发。© 2023韩国兽医科学学会。

Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied.This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection.PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity.The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner.We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.© 2023 The Korean Society of Veterinary Science.