**1. Introduction**

The diverse family of the *Coronaviridae* causes infections in a wide range of mammals, birds and humans. Feline coronavirus (FCoV) is a highly prevalent member of the *Coronaviridae* family and is found in both domestic and wild cat populations worldwide [1]. FCoVs occur in two different biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV infections commonly manifest as mild or asymptomatic infections of the feline enteric tract. Infections are often persistent and display intermittent shedding of virus over long periods of time which greatly contributes to the high seropositivity levels found in domestic cats. In single household cats, 20–60% of cats display signs of exposure to the virus and up to 90% of cats in multi-cat populations are seropositive [2–4].

FIPV emerges through mutations from the harmless FECV and can lead to a fatal clinical condition known as feline infectious peritonitis (FIP) [5–9]. Although the molecular pathogenesis of FIP is poorly understood [10–12], promising therapeutical approaches have recently been described [13–18]. It is

important to note that both biotypes exist in two serotypes [19–21]. Serotype II FCoVs are the results of recombination between a serotype I FCoV and a closely related canine coronavirus (CCoV) [22–25] and can easily be grown in vitro. In sharp contrast, the more relevant and prevalent serotype I FCoVs cannot be propagated in cell culture. Accordingly, serotype II viruses were often used in the past to gain insight into FCoV biology instead of serotype I FCoVs. To elucidate the molecular pathogenesis of FIP, cell culture-adapted serotype I FIPV laboratory strains were obtained over time [26]. However, these viruses proved to be unsuitable to study the pathogenesis of FIP due to the loss of pathogenicity via cell culture adaptation [1,9,26]. The first reverse genetic system that enabled genetic manipulation of the entire FCoV genome was described by Tekes et al. (2008) for serotype I FIPV laboratory strain Black using a vaccinia virus vector [26–28]. However, animal experiments showed that like many other laboratory strains, serotype I FIPV Black lost its capability to induce FIP [26]. On the contrary, another commonly used serotype II FIPV laboratory strain, 79-1146, [26,29,30] is much more pathogenic and thus does not appropriately resemble most of the field strains either. Due to the lack of suitable in vitro systems for field serotype I FECVs, it is critical to establish a suitable in vitro system that enables the growth of serotype I FECV. This culture system for serotype I FECV field viruses would not only provide insight into the molecular mechanism by which FECVs persist in the gu<sup>t</sup> for a longer period of time but it might also contribute to the understanding of how FIPV can evolve from FECV during a harmless persistent infection.

Over the past years, organoids have been employed as an in vitro system to support the growth of several human viruses that were unable to be cultured using standard cell culture methods [31,32]. Organoids are derived from either induced pluripotent stem cells (iPSCs) or from tissue-derived stem cells, which are grown and differentiated as three-dimensional structures that closely recapitulate the cellular composition and functions of their originating organ [33]. Tissue-derived organoids rely on the ability to isolate stem cell containing crypts. These crypts are then grown in the presence of differentiation factors (Wnt3a, R-Spondin, Noggin and EGF), allowing them to grow into three-dimensional mini-gut organoids [33]. As these complex cultures more closely resemble the multi-cell types found in their natural tissue counterparts, they often contain factors, which are required for the replication and propagation of viruses that are missing in standard cell cultures. To determine if these model systems could be used to support the relevant serotype I FECV growth, we established a cat intestinal organoid culture system and show that it is capable of supporting infection with GFP-expressing recombinant serotype I FECV generated by reverse genetics. This model will now open the doors to study the molecular mechanism of serotype I FECV-persistence in its natural enteric environment.

### **2. Materials and Methods**

### *2.1. Viruses and Cell Lines*

Serotype I recFECV-GFP and recFECV-S79-GFP were produced in vitro using the reverse genetic system for FCoV field strains described previously [34]. Recombinant virus stocks of recFECV-S79-GFP were titrated by plaque assay on routinely used felis catus whole fetus (FCWF) cells [26–28,35,36]. Virus stocks of recFECV-GFP which cannot be cultivated in standard cell culture systems were quantified by comparative Western blot analysis of the FCoV M protein together with recFECV-S79-GFP [34]. The cell line FCWF was provided by the diagnostic laboratory at the Justus Liebig University Giessen and maintained in culture media (DMEM with 1× penicillin/streptomycin (Thermo, Waltham, MA, USA) and 10% FBS (Biochrom, Cambridge, UK)). According to our experience with propagation of FCoV laboratory strains, the FCWF cells were used at a confluency of approximately 90% for the infection with recFECV-S79-GFP.
