**2. Materials and Methods**

#### *2.1. Cells and Reagents*

HEp-2 cells (BioWhittaker, Walkersville, MD, USA) were grown in minimum essential medium (MEM) supplemented with 10% of fetal calf serum (FCS), 1% of L-glutamine, 1% of nonessential amino acids, and 1% of penicillin and streptomycin. The human ductal cell line Panc-1 (ATCC) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of FCS, 1% of L-glutamine, and 1% of penicillin and streptomycin.

Fluoxetine chlorhydrate (Lilly France, Fegersheim, France) was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 5.48 uM and was used in all experiments, as previously reported [12]. Guanidine hydrochloride (GuHCl) was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France) and was used at a final concentration of 2 mM.

#### *2.2. Virus and Persistent Infection*

The diabetogenic CVB4 E2 strain, provided initially by Ji-Won Yoon (Julia McFarlane Diabetes Research Center, Calgary, Alberta, Canada), was propagated in HEp-2 cells and used to establish CVB4 persistent infections.

The model of persistent CVB4 infection of Panc-1 cells has been previously described [13,14]. Briefly, a 25 cm2 Nunc cell culture flask (Thermofisher Scientific, Villebon, France) containing an average of 106 cells in DMEM was inoculated with CVB4 at a multiplicity of infection (MOI) of 0.01. During the acute lytic infection, the culture medium was regularly changed, and finally a stable equilibrium was found between the viral replication and cell proliferation. The medium was changed twice a week, and cells were scraped and subcultured once a week. The supernatants were collected at different time points (1, 10, 20, 21, 24, 28, and 30 weeks post infection) during the persistent infection.

#### *2.3. Antiviral Activity Testing*

The antiviral activity of fluoxetine was evaluated using HEp-2 cells. Cells were seeded in a 96-well cell culture plate at 1.25 <sup>×</sup> 104 cells per well. Cells were inoculated with the virus at a MOI of 0.01, mixed with fluoxetine or DMSO. The plates were incubated at 37 ◦C, and the cell cultures were observed every day. The supernatants were collected when 100% cytopathic effect (CPE) was observed in DMSO-treated wells.
