Quantitative Genomic and Antigenomic Enterovirus RNA Detection in Explanted Heart Tissue Samples from Patients with End-Stage Idiopathic Dilated Cardiomyopathy

Lévêque, Nicolas; Renois, Fanny; Talmud, Déborah; Nguyen, Yohan; Lesaffre, François; Boulagnon-Rombi, Camille; Bruneval, Patrick; Fornès, Paul

doi:10.1128/jcm.01612-12

Cited by 26 publications

(32 citation statements)

References 17 publications

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“…These studies provided a possible link between the generation of noncytopathic viruses via genomic-RNA terminal deletions and the establishment and/or maintenance of persistent infections. Recently, similar deletions have been detected for the first time in cardiac biopsy specimens from patients suffering from chronic cardiomyopathy (29,36). These deletions, ranging from 15 to 48 nucleotides in length, involved a 5= RNA secondary structure (S-L I) that is the binding site for both viral and host cell proteins forming RNA replication complexes.…”

Section: Discussionmentioning

confidence: 93%

“…Among the terminally deleted CVB3 RNAs tested, RNA synthesis was observed only for those harboring deletions of either 7 or 21 nucleotides. The only RNAs produced from these deleted templates were double-stranded RNAs (dsRNAs) (without detectable single-stranded RNAs [ssRNAs]), suggesting an abnormal positive-strand/negative-strand RNA ratio near 1, similar to the ratios found in vivo for TD viruses (36).…”

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confidence: 91%

“…Indeed, the discovery of TD mutations in heart biopsy specimens was associated with low viral loads and abnormal positive-strand/ negative-strand viral RNA ratios, close to 1 rather than the high ratios (30 to 70) normally observed in enterovirus-infected cells with wild-type viruses (32,(36)(37)(38). Moreover, TD variants of CVB3 replicate in both cell culture and tissues from experimentally inoculated mice and from naturally infected humans without apparent cytopathic effect (31,32).…”

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confidence: 99%

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Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Lévêque

Garcia

Bouin

et al. 2017

J Virol

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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-

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Section: Discussionmentioning

confidence: 93%

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confidence: 91%

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confidence: 99%

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Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Lévêque

Garcia

Bouin

et al. 2017

J Virol

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“…Full-length CVB3-Nancy RNA transcripts containing the target sequences were synthesized and used as a positive control for copy number calculation. Sensitivity of the PCR assay was estimated to 100 copies per PCR well reaction by 10-fold serial limit dilutions of these RNA transcripts (11).…”

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confidence: 99%

Enterovirus 68 in Pediatric Patients Hospitalized for Acute Airway Diseases

Renois¹,

Bouin²,

Andréoletti³

2013

J Clin Microbiol

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Enterovirus 68 was detected in 10 respiratory specimens from pediatric patients hospitalized for acute wheezing or bronchitis during 2009 in the northeast of France. Viral loads ranged from 2 ؋ 10 5 to 7.2 ؋ 10 7 copies/ml. Alignment of 5= nontranslated regions and phylogenetic analysis of partial VP1 gene sequences show that these viruses clustered and belonged to clade C. Human enterovirus 68 (HEV-D68) was isolated from respiratory specimens for the first time in 1962 in the United States in children with pneumonia and bronchiolitis (1). Unlike other enteroviruses, HEV-D68 is acid labile and biologically more similar to human rhinoviruses in being mainly associated with respiratory diseases; however, until recently, reports of respiratory infections due to HEV-D68 were rare (2, 3). Over the past 3 years, outbreaks in Japan, the Philippines, and the Netherlands as well as epidemic clusters in the United Kingdom have implicated HEV-D68 as an emerging respiratory pathogen (2, 4-9). The clinical presentation of HEV-D68 infections during these outbreaks ranged from mild illness to rare severe pneumonia cases requiring admission in intensive care units (2,(4)(5)(6)(7)(8)(9). Among these epidemic strains, novel genetic variants were described (2, 4-8). The present classical enterovirus molecular assays used in routine virological diagnosis can fail to detect HEV-D68 strains in respiratory samples, leading to an underestimation of the prevalence and the role of HEV-D68 infections in pediatric acute airway diseases (10). We report here the phylogenetic characterization of HEV-D68 strains detected in a cohort of pediatric patients hospitalized for acute airway diseases.The study. Total nucleic acid extraction was performed using the NucliSens EasyMAG instrument (bioMérieux, Lyon, France), according to the manufacturer's instructions. A quantitative realtime reverse transcription (RT)-PCR targeting the 5= nontranslated region (5=NTR) of the Enterovirus genus (EV; including enteroviruses HEV A to D and rhinoviruses HRV A to C) was carried out to perform a pan-enterovirus and rhinovirus detection and to quantify the viral load. Briefly, 2 l total nucleic acid extract was amplified in a 10-l RT-PCR mixture containing 5 l 2ϫ reaction mix, 2 U SuperScript III RT/Platinum Taq mix (Invitrogen, Life Technologies, Saint-Aubin, France), and 0.4 M each primer and probe. The primer sequences used were 5=-AGCCTG CGTGGCKGCC-3= and 5=-GAAACACGGACACCCAAAG-3=, and the probe was 5=-6-carboxyfluorescein (FAM)-CTCCGGCC CCTGAATGYGGCTAA-3= (10). The cycling conditions included initial incubations at 55°C for 30 min and 94°C for 2 min, followed by 40 cycles of 94°C for 15 s and 63°C for 1 min. Full-length CVB3-Nancy RNA transcripts containing the target sequences were synthesized and used as a positive control for copy number calculation. Sensitivity of the PCR assay was estimated to 100 copies per PCR well reaction by 10-fold serial limit dilutions of these RNA transcripts (11).To discriminate HEV from HRV strains, we performed a specific HEV...

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“…To gain insight into the pathogenesis of virus-associated DCM, the detection of a broad panel of virus species combined with the measurement of the viral load in heart tissue is required to assess the viral epidemiology and to distinguish active from chronic or persistent cardiac viral infections (7). Virological diagnosis of acute or chronic cardiomyopathies is currently based on combinations of quantitative real-time PCR (RT-qPCR) assays (1,8). However, the wide range of viruses potentially responsible for cardiac infections, including both DNA and RNA viruses, with their highly various genetic characteristics, renders rapid and exhaustive virological diagnosis difficult using multiple monoplex RT-PCR assays (3).…”

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Virus Detection and Semiquantitation in Explanted Heart Tissues of Idiopathic Dilated Cardiomyopathy Adult Patients by Use of PCR Coupled with Mass Spectrometry Analysis

Nguyen¹,

Renois²,

Lévêque³

et al. 2013

J Clin Microbiol

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Quantitative Genomic and Antigenomic Enterovirus RNA Detection in Explanted Heart Tissue Samples from Patients with End-Stage Idiopathic Dilated Cardiomyopathy

Cited by 26 publications

References 17 publications

Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Enterovirus 68 in Pediatric Patients Hospitalized for Acute Airway Diseases

Virus Detection and Semiquantitation in Explanted Heart Tissues of Idiopathic Dilated Cardiomyopathy Adult Patients by Use of PCR Coupled with Mass Spectrometry Analysis

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