Our findings demonstrate a significantly higher proportion of active coxsackievirus B cardiovascular infections in patients who suddenly died of MI compared with matched control subjects, suggesting that these EVs may significantly contribute to the pathogenesis of acute MI by a focal disruption of the dystrophin-glycoprotein complex.

Enterovirus RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.

Our findings evidence an EV-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis. Moreover, our results indicate that this EV-induced pro-apoptotic mechanism could be partly related to an up-regulation of Bax expression, and suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Enteroviruses and other cardiotropic viruses have been associated with the development of late severe adverse cardiac events in infants receiving heart transplants. However, the source and the chronology of cardiac allograft infection by an enterovirus in patients receiving heart transplants remain unknown. Using RT-PCR and immunohistochemistry assays, endomyocardial tissue samples of 30 adult patients were tested to detect the presence of specific enterovirus 5' non-coding (5'NC) sequences and of VP1 capsid protein, and this at the time of cardiac transplantation and at the 12-month biopsy for graft rejection control. Moreover, the endomyocardial detection of genomic sequences of enteroviruses, Epstein-Barr virus, herpes simplex virus, cytomegalovirus (CMV), varicella-zoster virus, adenoviruses, and parvovirus B19 was carried out by RT-PCR and polymerase chain reaction (PCR) assays at the time of late severe cardiac events. Enterovirus RNA and VP1 antigen were both detected in 4 (13%) of 30 patients at the time of the 12-month biopsy for graft rejection control, whereas no enterovirus component was detected in the explanted and implanted heart tissues taken from these 4 patients at the time of transplantation. At the time when severe cardiac events were developed, within 3 months after the positive enterovirus cardiac detection, these four patients demonstrated the presence of endomyocardial enterovirus RNA sequences whereas they were tested negative for the endomyocardial detection of genomic sequences from DNA viruses (except for CMV in two cases), and for a significant level of pp65 CMV antigenemia. Taken together, these findings indicate that enteroviruses could be acquired as a new endomyocardial infection within 12 months after transplantation in adults receiving heart transplants, and suggest that this infection might be an etiological cause for unexplained late severe adverse cardiac events in the heart-transplantated adults.