J Vet Sci.  2023 Jul;24(4):e53. 10.4142/jvs.23017.

Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection

• Lee SA
• Lee HJ
• Gu NY
• Park YR
• Kim EJ
• Kang SJ
• Hyun BH
• Yang DK

• KMID: 2545100
• DOI: http://doi.org/10.4142/jvs.23017

Abstract

Background
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 swineMRV3 has rarely been studied.
Objectives
This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection.
Methods
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 threedimensional (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 realtime reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity.
Results
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.
Conclusions
We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

Keyword

Mammalian orthoreovirus 3; organoid; in vitro techniques, jejunum