Emergence of Fluoxetine-Resistant Variants during Treatment of Human Pancreatic Cell Cultures Persistently Infected with Coxsackievirus B4

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: coxsackievirus B4; enteroviruses; fluoxetine; mutations; persistent infection; resistance.

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.

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.

Figure 3

Amino-acid substitutions in fluoxetine-resistant virus. Virus suspensions were collected from persistent CVB4 E2-infected cultures at baseline, and after 4 and 10 weeks of treatment with DMSO or fluoxetine. The sequence of the whole CVB4 E2 2C region (from nt 4039 to nt 5025, 329 aa) was determined using Sanger method. The amino-acid substitutions in the sequences of fluoxetine-resistant viruses (I3 and I4) are shown.