Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses

Abstract

Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79–1683 and WSU 79–1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79–1683 still replicated significantly more efficient compared to FCoV WSU 79–1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

“…Although PoCo83-3 cells maintained expression of the epithelial marker KRT18 up to P43, we observed the expression of VIM, commonly described as a marker for mesenchymal cells and epithelial-mesenchymal transition [Lee et al, 2006;Satelli and Li, 2011]. However, VIM expression in epithelial cells has also been detected in primary cultures [Rusu et al, 2005;Stumpff et al, 2011] and in other epithelial cell lines [Loret et al, 2009;Anand et al, 2012;Desmarets et al, 2013;Zakrzewski et al, 2013]. It has been proposed that VIM expression in epithelial cells may represent an essential adaption mechanism to the artificial environment during in vitro growth [Mark et al, 1990;Baer and Bereiter-Hahn, 2012].…”

“…However, some authors reported that hTERT alone was not sufficient to immortalize some epithelial cell types, like keratinocytes or mammary epithelial cells [Kiyono et al, 1998], and that the p16(INK4A)-pRb pathway might play an important role here [Kim et al, 2002;Rheinwald et al, 2002]. Furthermore, some work groups used a combination of SV40 TAg and hTERT to generate immortalized cell lines [Matsumura et al, 2004;Desmarets et al, 2013;Olyslaegers et al, 2013]. In previous studies using primary pCECs [Petto et al, 2011;Lesko et al, 2013], these cells seemed to be quite sensitive when subjected to cell culture, usually maintaining active proliferation for only a few passages (according to our own unpublished observations).…”

“…A contamination by mesenchymal cells is a common problem in pCEC cultivation [Petto et al, 2011] and epithelial cell culture establishment in general [Anand et al, 2012;Desmarets et al, 2013]. To generate a homogenous cell line possessing consistent and reproducible characteristics, we selected clonal epithelial cell clusters to ensure that the PoCo83-3 cell line was derived from a single epithelial cell clone [Loret et al, 2009;Miyazawa et al, 2010].…”

“…Consequently, researchers are forced to frequently reestablish cultures, but this is often restricted for ethical and financial reasons [Vos et al, 2012]. Moreover, the standardization of primary cell culture protocols is difficult due to a high variability between individual isolations, rapid dedifferentiation [Hartung et al, 2002], and a high incidence of fibroblastic overgrowth [Barnes and Masui, 1981;Jensen and Therkelsen, 1982;Katayama et al, 1984;Desmarets et al, 2013]. These limitations, along with the need for sufficient and consistent sample materials, have increased the demand for well-characterized and continuously growing cell lines, particularly in studies of epithelial barrier function where the availability of adequate cell culture models is essential.…”

“…A common and reliable approach towards overcoming cellular senescence is the use of simian virus 40 large T antigen (SV40 TAg) expression constructs [Telgmann, 2001;Kirchhoff et al, 2004;Loret et al, 2009;Miyazawa et al, 2010;Desmarets et al, 2013;Gleich et al, 2016;Hu et al, 2016;Kim et al, 2016]. A complex signaling network involving the p53 and p16(INK4A)-pRb tumor suppressor pathways plays a predominant role in "replicative senescence" [Hara et al, 1991;Hahn, 2002].…”