*3.2. Investigation of Resistance to Fluoxetine Treatment*

To confirm that I3 and I4 were resistant to the treatment, the susceptibility of viral suspensions to fluoxetine was evaluated in a model of acute infection. Various viral suspensions from persistently infected cultures treated with fluoxetine at 5.48 μM or DMSO were collected at week 8 o.t. They were inoculated into HEp-2 cells in the presence of fluoxetine or DMSO. Supernatants were collected on day 3 p.i., and the viral titers were determined. Figure 2 presents the extent of decrease in viral titer in fluoxetine-treated wells, as compared to DMSO-treated ones.

**Figure 2.** Susceptibility to fluoxetine of virus isolates obtained from treated persistently CVB4-infected cultures. Virus suspensions were collected from persistent CVB4 infections treated with DMSO (I1-D, I2-D, I3-D, and I4-D), or with fluoxetine (I3-F and I4-F) at week 8 of treatment. HEp-2 cells were inoculated with various virus suspensions in the presence of fluoxetine or dimethyl sulfoxide (DMSO). Viral titers were determined 3 days postinoculation. Data are mean ± SD of two independent experiments.

Virus isolates emerging from the four persistently infected cultures treated with DMSO for eight weeks (I1-D, I2-D, I3-D, and I4-D) remained highly susceptible to fluoxetine. Indeed, a mean decrease in viral titer ranging between 4 and 4.5 log TCID50/mL was observed when the isolates were inoculated into HEp-2 cell cultures in the presence of fluoxetine, as compared to the cultures inoculated with virus isolates in the presence of DMSO.

Regarding the virus obtained from persistently infected cultures treated with fluoxetine for 8 weeks, the antiviral activity of fluoxetine was strongly reduced. The mean decrease in viral titer was as low as 0.1 and 1.2 log TCID50/mL for I3-F and I4-F, respectively (see Figure 2).

No viral titer was obtained in persistently infected cultures I1 and I2 treated with fluoxetine (I1-F and I2-F) after 4 weeks of treatment; therefore, they were not tested.

Overall, the mean decrease in viral titer was significantly lower when cells were inoculated with the virus isolated from persistently infected cultures treated with fluoxetine than those from persistently infected cultures treated with DMSO (0.6 log TCID50/mL versus 4.2 log TCID50/mL, *p* < 0.0001).

The same experiments were run using GuHCl instead of fluoxetine. When the viral suspensions were inoculated into HEp-2 cells in presence of GuHCl, a mean decrease of 4.7 log TCID50/mL and 5 log TCID50/mL was observed when cells were inoculated with the virus obtained from persistently infected cultures treated with DMSO and persistently infected cultures I3-F and I4-F treated with fluoxetine, respectively for 8 weeks. There is no statistical difference between these viral titer reductions.
