Viral infections are described as environmental factors that are implicated in various thyroid diseases. The role of enteroviruses (EV) in the pathogenesis of thyroid diseases has been suspected. Recently, we found that EV RNA could be detected in postoperative thyroid specimens. We decided to investigate the infection of a human thyroid cell line with coxsackievirus B4 (CVB4).The wild-type human thyroid carcinoma cells K1 were inoculated with CVB4E2 at 2.1 x 10(7) TCID50/mL. The titer of the virus progeny was determined every 3 days on HEp-2 cells. CVB replication at the molecular level was monitored by searching for intracellular viral genomes using reverse transcription (RT)-polymerase chain reaction (PCR). EV VP1 capsid protein was detected by indirect immunocytofluorescence staining. Cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) absorbance, and the nuclear morphology was assessed by Hoechst Dye staining.Infectivity assays with CVB4E2 revealed an increase in viral titers. Virus production decreased thereafter, but was not stopped by serial subculture for 24 days after infection. Detection of intracellular positive and negative viral RNA strands by RT-PCR was positive between days 1 and 14 postinfection (p.i.), and by semi-nested RT-PCR up to 24 days. K1 cell cultures infected with CVB4 were stained positively for EV VP1: the number of VP1-positive cells decreased rapidly within 6 days and remained low up to the end of culture. Compared with mock-infected cultures, viability in CVB4-infected cultures was around 100% up to 24 days. Cells with strongly fluorescent nuclei and/or fragmented nuclei were observed.We demonstrate for the first time that CVB4 could replicate in thyroid cells and could persist, with predominance of viral negative RNA strands for up to 24 days p.i. without obvious cytopathic effect. Our results suggest that CVB4 may lead to thyroid cell apoptosis. Further studies are needed to determine whether CVB could play a role in thyroid pathologies.