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Abstract

Intestinal epithelial cells (IECs) line the surface of the intestinal epithelium and act as a barrier against commensal microbiota. When enteric viruses infect IECs, they induce the production of two types of cytokines, type-I and type-III interferons (IFNs), which can set an antiviral state in the tissue. One of the main pathogens of the intestines is rotavirus, which was shown to elicit the upregulation of both types of IFNs in mice models and commercial cell lines. Nevertheless, the role that each type of IFN plays during rotavirus spread has not been thoroughly evaluated. Here, I generated a collection of fluorescent tools to evaluate rotavirus infection and spread in different contexts using live cell fluorescence microscopy. I could show that rotavirus efficiently blocks type-I IFN-mediated upregulation of interferon stimulated genes (ISGs) through its NSP1 protein, and only type-III IFNs can be upregulated. Moreover, even in the absence of NSP1, only type-III IFNs were able to rapidly establish an antiviral state in a large number of IECs and prevent the spread of rotavirus. On the contrary, type-I IFNs had a delayed antiviral effect, which allowed infection of IECs by newly produced viruses. Moreover, I could observe that rotavirus infection is strongly dependent on ADP-mediated calcium waves. Live microscopy experiments showed that second rounds of infection seem to take place in areas defined by calcium waves elicited by the first round of infection. Importantly, blocking of ADP signaling through the P2Y1 purinergic receptor prevented new infections from taking place, highlighting the importance of calcium waves during rotavirus infection. In conclusion, my results suggest that only type-III IFNs are able to control rotavirus infection and spread in IECs, and that type-I IFNs do not seem to play a role at least in the antiviral state of the intestinal epithelium. Furthermore, calcium waves generated by the first round of infection likely alter the integrity of IECs to allow viruses to more easily infect the culture. I propose that IECs can efficiently control the spread of enteric viruses due low amounts of type-III IFNs being needed to set an antiviral response in a notably high number of cells, and that rotavirus-induced calcium waves alters the polarized nature of IECs to facilitate infection.