Background: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. Methods: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5’ termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. Results: Major enterovirus B populations characterized by 5’ terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5’ genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. Conclusions: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5’ terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

Diarrhea is a frequent complication after kidney transplantation, ascribed to adverse effects of the immunosuppressive therapy in case of negative microbiological examination of the stools. The aim of this study was to improve the microbiological diagnosis by implementing molecular tests. Fifty-four severe diarrhea events that occurred in 49 adult kidney transplant recipients from September 2010 to November 2011 were investigated. One or several enteric pathogens were detected in 13 (23%) stool samples using classical microbiological methods versus 39 (72%) for the seven commercially available multiplex PCR assays used retrospectively (P ‫؍‬ 0.006). Interestingly, molecular diagnosis identified 15 multiple infections compared to none using classical techniques. The primary pathogens detected were enteropathogenic Escherichia coli (EPEC) (n ‫؍‬ 15; 38%), Campylobacter spp. (n ‫؍‬ 15; 38%), and Norovirus (n ‫؍‬ 14; 36%). Specificities for Campylobacter and Norovirus infection diagnosis were 75 and 100%, respectively, by comparison to reference methods. Based on molecular findings, a cyclosporine-mycophenolate mofetil combination was identified as a risk factor for developing Norovirus-induced diarrhea. Norovirus infections were also responsible for higher weight loss than all the other causes of diarrhea. In samples from asymptomatic immunocompromised and immunocompetent patients, EPEC but not Norovirus and Campylobacter infections were detected at a frequency similar to that observed in symptomatic kidney transplant recipients. In conclusion, molecular tools significantly improved the detection of single and multiple enteric infections by comparison to classical techniques and could quickly become the key element in the management of severe acute diarrhea in transplant recipients.

Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5=-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5= stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5= deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5= deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7-and 21-nucleotide deletions. Since 5= stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CD pro , to deleted positive-strand RNAs corresponding to the 5= end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CD pro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5=-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5= end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5=-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis as-

We performed deep sequencing analysis of the enterovirus 5′ noncoding region in cardiac biopsies from a patient with dilated cardiomyopathy. Results displayed a mix of deleted and full-length coxsackievirus B3, characterized by a low viral RNA load (8.102 copies/μg of nucleic acids) and a low viral RNA positive-sense to RNA negative-sense ratio of 4.8.