Establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses

Tingting Cui,Sebastiaan Theuns,Gaëtan M A De Gryse,Lowiese M B Desmarets,Jiexiong Xie,Hans J Nauwynck,Bo Yang,Wim Van Den Broeck

doi:10.1038/s41598-018-33305-1

Abstract

A stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. Because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. As subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. First, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain RVA/pig-tc/BEL/RV277/1977/G1P[7] and different other rotavirus genotypes (fecal samples containing G5P[7], G5P[13], G9P[23], G4P[6]). Next, the TGEV Purdue strain infected both ileocytes and colonocytes whereas the Miller strain only infected ileocytes. Last, the PEDV CV777 Vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. The infectivity of the CV777 Vero adapted strain was higher when the cells were cultured without fetal bovine serum and the CV777 fecal suspension only infected the ileocytes cultured without fetal bovine serum. In conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses.

Highlights

Enteric viruses are common causes of diarrhea in humans and animals
A myofibroblast cell line was established from porcine ileum which was identified by the presence of α-smooth muscle actin, vimentin and fibronectin
In the absence of myofibroblasts, epithelial cells died after 2–3 days, even when supplemented with 20% conditioned medium collected from myofibroblast cultures

Summary

Introduction

Enteric viruses are common causes of diarrhea in humans and animals. Porcine rotavirus, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are well known enteric viruses, leading to high morbidity and mortality in piglets and causing economic losses in swine-producing countries. Transmissible gastroenteritis virus and porcine epidemic diarrhea virus belong to the group I coronaviruses They are enveloped viruses with an approximately 28.5 kb single-stranded, positive-sense RNA genome encoding four structural proteins: the spike (S), membrane (M), envelope (E), and nucleoprotein (N) protein. Like TGEV and PEDV, rotavirus is mainly transmitted by fecal-oral route and virus infection causes the destruction of mature small intestinal enterocytes. Most rotavirus research was conducted on non-polarized MA104 cells (African green monkey kidney epithelial cells) which are easy to culture and permissive for certain rotavirus strains of different genotypes. Data concerning the replication cycles of these enteric viruses in their target cell (mature intestinal enterocytes) are scarce. To this end, it is essential to obtain cultures of porcine intestinal enterocytes. To determine the usability of this co-culture system, enteric rotaand coronaviruses were used to infect the co-cultured enterocytes

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Conclusion