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Group B Coxsackieviruses (CV-B) are responsible for various acute human diseases, and they are involved in chronic diseases such as type 1 diabetes. It has been reported that fluoxetine (FLX) inhibited CV-B4E2 in human cell lines in vitro. In so far as CV-B4E2 can replicate in CD1 mice, it was investigated whether FLX could inhibit CV-B4E2 in vitro and in vivo in mouse systems. When 5.5 μM FLX was added to CV-B4E2-infected Min-6 cell (murine pancreas beta cell line) cultures, the virus-induced cytopathic effect was inhibited. In this system and in CV-B4E2-infected CD1 mouse pancreatic organotypic cultures treated with FLX the levels of infectious particles in supernatant fluids were below the limit of detection of the assay. The administration of FLX (10 mg/kg/day) by intraperitoneal route resulted in significant reduced levels of infectious particles in heart and pancreas of mice inoculated with CV-B4E2 by the same route. In conclusion FLX can inhibit CV-B4 in vitro and in vivo in mouse systems, additional studies are needed to investigate further the potential value of FLX to combat CV-B4 infections and to treat CV-B4-induced diseases.Fluoxetine inhibits the replication of CV-B4E2 in a mouse beta cell line.Fluoxetine inhibits the replication of CV-B4E2 in mouse pancreatic organotypic culture.Fluoxetine can reduce the level of infectious particles in organs of mice infected with CV-B4E2.