*3.1. Persistent CVB4 Infection and Fluoxetine Treatment*

Four independent cultures persistently infected with CVB4 (I1, I2, I3, and I4) were established in Panc-1 cells. Culture supernatants were periodically collected and the presence of infectious particles was checked using HEp-2 cells. The viral titers obtained in the supernatants ranged from 6.83 to 7.83 log TCID50/mL at one week postinoculation (p.i.), 6.25 to 7.50 log TCID50/mL at ten weeks p.i., and 5.50 to 6.50 log TCID50/mL at twenty weeks p.i. (see Figure 1).

**Figure 1.** Persistent coxsackievirus B4 (CVB4) infection in Panc-1 cells and treatment with fluoxetine. Four independent persistent CVB4 infections (I1, I2, I3, and I4) were established in Panc-1 cells. At 20 weeks post infection, cells were treated with fluoxetine at 5.48 μM twice a week. The culture supernatants were collected all along the follow-up. Viral titers in supernatants were determined using the end-point dilution assay.

Starting from week 20 p.i., persistently infected cultures were treated twice a week with fluoxetine at 5.48 μM or DMSO, and supernatants were collected periodically to monitor the infection. The levels of infectious viral particles in persistently infected cultures I1 and I2 decreased significantly at one week of treatment (o.t.) i.e., 2.5 log TCID50/mL, and the virus was undetectable at four weeks posttreatment. In contrast, for persistently infected cultures I3 and I4, only a slight decrease was observed in viral titers at one week o.t., followed by a rise from the fourth week o.t. The viral titers obtained after 10 weeks of fluoxetine-treatment were still at 6.5 and 6.25 TCID50/mL for I3 and I4, respectively (see Figure 1). No significant changes in viral titers were observed in DMSO-treated cultures.
