**Enagnon Kazali Alidjinou, Antoine Bertin, Famara Sane, Delphine Caloone, Ilka Engelmann and Didier Hober \***

Faculté de médecine, Université Lille, CHU Lille, Laboratoire de Virologie EA3610, F-59000 Lille, France; enagnonkazali.alidjinou@chru-lille.fr (E.K.A.); antoine.bertin@gmail.com (A.B.); famara.sane@chru-lille.fr (F.S.); delphine.lobert@univ-lille.fr (D.C.); ilka.engelmann@chru-lille.fr (I.E.)

**\*** Correspondence: didier.hober@chru-lille.fr; Tel.: +33-0-3-20-44-66-88; Fax: +33-03-20-44-48-95

Received: 10 April 2019; Accepted: 21 May 2019; Published: 28 May 2019

**Abstract:** This study reports the antiviral activity of the drug fluoxetine against some enteroviruses (EV). We had previously established a model of persistent coxsackievirus B4 (CVB4) infection in pancreatic cell cultures and demonstrated that fluoxetine could clear the virus from these cultures. We further report the emergence of resistant variants during the treatment with fluoxetine in this model. Four independent persistent CVB4 infections in Panc-1 cells were treated with fluoxetine. The resistance to fluoxetine was investigated in an acute infection model. The 2C region, the putative target of fluoxetine antiviral activity, was sequenced. However, Fluoxetine treatment failed to clear CVB4 in two persistent infections. The resistance to fluoxetine was later confirmed in HEp-2 cells. The decrease in viral titer was significantly lower when cells were inoculated with the virus obtained from persistently infected cultures treated with fluoxetine than those from susceptible mock-treated cultures (0.6 log TCID50/mL versus 4.2 log TCID50/mL, *p* < 0.0001). Some previously described mutations and additional ones within the 2C protein were found in the fluoxetine-resistant isolates. The model of persistent infection is an interesting tool for assessing the emergence of variants resistant to anti-EV molecules. The resistance of EV strains to fluoxetine and its mechanisms require further investigation.

**Keywords:** enteroviruses; coxsackievirus B4; persistent infection; fluoxetine; resistance; mutations
