Establishment of an In Vitro Model to Study Viral Infections of the Fish Intestinal Epithelium

Abstract

Viral infections are still a major concern for the aquaculture industry. For salmonid fish, even though breeding strategies and vaccine development have reduced disease outbreaks, viral diseases remain among the main challenges having a negative impact on the welfare of fish and causing massive economic losses for the industry. The main entry port for viruses into the fish is through mucosal surfaces including that of the gastrointestinal tract. The contradictory functions of this surface, both creating a barrier towards the external environment and at the same time being responsible for the uptake of nutrients and ion/water regulation make it particularly vulnerable. The connection between dietary components and viral infections in fish has been poorly investigated and until now, a fish intestinal in vitro model to investigate virus–host interactions has been lacking. Here, we established the permissiveness of the rainbow trout intestinal cell line RTgutGC towards the important salmonid viruses—infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (subtype 3, SAV3) and infectious salmon anemia virus (ISAV)—and explored the infection mechanisms of the three different viruses in these cells at different virus to cell ratios. Cytopathic effect (CPE), virus replication in the RTgutGC cells, antiviral cell responses and viral effects on the barrier permeability of polarized cells were investigated. We found that all virus species infected and replicated in RTgutGC cells, although with different replication kinetics and ability to induce CPE and host responses. The onset and progression of CPE was more rapid at high multiplicity of infection (MOI) for IPNV and SAV3 while the opposite was true of ISAV. A positive correlation between the MOI used and the induction of antiviral responses was observed for IPNV while a negative correlation was detected for SAV3. Viral infections compromised barrier integrity at early time points prior to observations of CPE microscopically. Further, the replication of IPNV and ISAV had a more pronounced effect on barrier function than SAV3. The in vitro infection model established herein can thus provide a novel tool to generate knowledge about the infection pathways and mechanisms used to surpass the intestinal epithelium in salmonid fish, and to study how a virus can potentially compromise gut epithelial barrier functions.

The effects of Pediococcus acidilactici MA18/5M on growth performance, gut integrity, and immune response using in vitro and in vivo Pacific salmonid models

“…Recent reports on a salmonid intestinal epithelial cell line derived from rainbow trout (O. mykiss), RTgutGC, have established the usefulness of this cell line as a model for functional studies on fish feed development based on gut barrier function and immune competence, as well as host-microbe interactions with viruses (36)(37)(38)(39)(40). Furthermore, by employing semipermeable membrane supports (Transwell ® ), it is possible to recapitulate the intestinal environment in vitro and conduct studies on the permeability and integrity of the cell monolayer (41).…”

Evaluation of rainbow trout (Oncorhynchus mykiss) organotypic intestinal platforms: cellular responses after long-term exposure to in vitro digested feed

“…To date, fish cell-based models are already used to study different biological processes and provide a substantial contribution in a variety of areas such as biomedical research, biotechnology, disease control, environmental toxicology, ecotoxicology, fish immunology, and virology (Wang et al, 2019;Goswami et al, 2022;Løkka et al, 2023). Moreover, fish cell lines easily adapt to a wide spectrum of temperature, are characterized by higher resilience to hypoxia conditions, and are easier to maintain for prolonged periods of time in culture compared to mammalian cell lines (Goswami et al, 2022).…”

Development of a Rainbow Trout (Oncorhynchus mykiss) Intestinal In Vitro Platform for Profiling Amino Acid Digestion and Absorption of a Complete Diet

“…Although the vast majority of the studies have been performed in mammals, several studies have focused on fish-related models, with rainbow trout (Oncorhynchus mykiss) as a target species due to available cell lines. The first fish intestinal cell line, named RTgutGC, was derived from this species several years ago [6] and has been extensively studied since, for various purposes, including toxicology [7][8][9], gut immunology [10], and intestinal function [11][12][13].…”