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(Circulation. 2000;101:231.)
© 2000 American Heart Association, Inc.

Brief Rapid Communications

Enteroviral Capsid Protein VP1 Is Present in Myocardial Tissues From Some Patients With Myocarditis or Dilated Cardiomyopathy

Yanwen Li, MD, PhD; Thomas Bourlet, PharmD; Laurent Andreoletti, PharmD, PhD; Jean-François Mosnier, MD, PhD; Tianqing Peng, MB, MM; Yingzhen Yang, MD; Leonard C. Archard, PhD, FRCPath; Bruno Pozzetto, MD, PhD; Hongyi Zhang, MB, PhD

From the Molecular Pathology Section, Division of Biomedical Sciences (Y.L., L.C.A., H.Z.) and Infectious Disease and Medical Microbiology (H.Z.), Imperial College of Science, Technology, and Medicine, London, UK; Laboratoire de Bactériologie-Virologie (Y.L., T.B., B.P.) and Laboratoire d’Anatomo-Pathologie (J.-F.M.), Faculté de Médecine Jacques Lisfranc, Saint-Etienne, and Laboratoire de Virologie, Faculté de Médecine, Lille (L.A.), France; and Key Laboratory of Viral Heart Disease, Ministry of Public Health, Zhongshan Hospital, Shanghai Medical University, PRC (Y.L., T.P., Y.Y.).

Correspondence to Dr Hongyi Zhang, Molecular Pathology, DBS, SAF Building, ICSTM, Exhibition Road, London SW7 2AZ, UK. E-mail h.zhang{at}ic.ac.uk

	Abstract

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Background—There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence.

Methods and Results—Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group–specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription–nested polymerase chain reaction, correlating with antigen detection (=0.6±0.21). Neither viral antigen nor RNA was detected in any controls.

Conclusions—Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Key Words: cardiomyopathy • myocarditis • viruses • antigens • immunohistochemistry

	Introduction

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Enteroviruses are the most common agents of myocarditis and have been implicated in the pathogenesis of dilated cardiomyopathy (DCM).¹ In a productive replication cycle, the virus entry is followed by an uncoating process leading to release of single-stranded, positive sense genomic RNA. Viral proteins are synthesized as a polyprotein, subsequently cleaved to produce the 4 capsid proteins VP1 through VP4 and nonstructural proteins necessary for virus replication. The epitopes for neutralizing antibodies are located predominantly in VP1 and are serotype-specific. There are also epitopes common to the enterovirus group in VP1, and antibodies against these determinants are heterotypic and nonneutralizing.²

The association between enterovirus infection and myocardial disease has been based on virus isolation, serological studies, nucleic acid hybridization, and reverse transcription–polymerase chain reaction (RT-PCR). There has been disparity in the detection of enteroviral RNA in the heart by RT-PCR.³ In the studies in which virus was detected in the heart,^{4 5} there was no evidence to demonstrate the localization of the virus. Previous histochemical demonstration of enteroviral antigens in myocardium has been limited, but recently, an enterovirus group–specific monoclonal antibody (mAb) has become available. This antibody reacts with a nonneutralizing epitope conserved on the capsid protein VP1 of a panel of enterovirus serotypes.⁶ In the present study, we used this reagent in an improved immunohistochemical technique to detect enteroviral antigen in myocardial sections and compared this with RT–nested PCR (RT-NPCR) already established in our laboratory.⁵

	Methods

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Patients
Explanted or postmortem myocardial samples were obtained from 19 patients with myocarditis or DCM histologically proven according to the Dallas criteria and those described previously¹ and from 11 controls. Patient details, including age, sex, and origin, are listed in the Table. Eight of 10 patients with myocarditis died within 1 week and the remaining 2 within 2 weeks after onset. The patients were from China (Shanghai or Chuxiong) or France (St Etienne or Lille) and were thought to have a history of virus infection. Chuxiong is a region with endemic cardiomyopathy associated with selenium deficiency and enterovirus infection. Ventricular tissue was obtained within 24 hours of death or transplantation, fixed in 10% neutral buffered formalin (3 samples fixed in Bouin’s solution), and paraffin-embedded. Consent was obtained from patients or their relatives.

View this table: [in this window] [in a new window]   Table 1. List of Patients: Detection of Enterovirus (EV) in Myocardial Tissue Samples

Improved Immunohistochemistry
The enterovirus group–specific mAb 5-D8/1 (IgG2a), normal mouse IgG2a, blocking reagent, antibody diluent, and the detection system EnVision/HRP were purchased from Dako Ltd. Heat-mediated antigen retrieval procedures were used.⁷ Immunohistochemistry with the polymer/peroxidase conjugate (EnVision) was performed according to published protocols.⁸ The mAb was applied at a dilution of 1:500 (0.22 µg/mL). As a control, the primary antibody was replaced with diluent only or concentration-matched mouse IgG2a.

Detection of Enteroviral RNA
Embedded myocardial samples were dewaxed with xylene and washed with ethanol. Total RNA was extracted in Tri reagent (Sigma Chemicals). Amplification of enteroviral RNA or a housekeeping gene was performed as described previously.⁵

	Results

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Presence of Enteroviral VP1 in Myocardial Tissue Sections From Patients
Enteroviral capsid protein VP1 was detected in duplicate myocardial tissue sections in 9 of 11 patients with myocarditis (81.8%) and 6 of 8 patients with DCM (75%) (Table). Signals were reproducible when further sections were tested and localized in the cytoplasm of myocytes (Figure). In contrast, all 11 controls were negative. Two immunostaining patterns were observed: focal or extensive staining of VP1 involving a large area of sections was observed in fatal myocarditis (Figure, A), whereas VP1 was detected in scattered myofibers in DCM involving a small part of the sections (Figure, B and C). In some cases, only a few myocytes were positive in an entire section, suggesting a possible correlation between patterns of virus detection and disease stage or severity. VP1-positive myocytes were found adjacent to dying myocytes or chronic inflammatory lesions in myocarditis. No signals were obtained when consecutive sections were incubated with mouse IgG2a or diluent only (Figure, D).

View larger version (122K): [in this window] [in a new window]   Figure 1. Detection of enterovirus capsid protein VP1 in myocardial tissue sections by immunohistochemistry. A, Immunostaining (yellow-brown) of VP1 in heart with fatal myocarditis (case 9). B1 and B2, Scattered immunostaining of VP1 in heart with DCM (case 14). C1 and C2, Viral antigens are localized in cytoplasm of myocytes adjacent to necrosis or inflammatory lesions in heart with chronic myocarditis (case 6). D, Serial section of C probed with normal mouse IgG2a, showing clear background and negative result. All sections were counterstained with hematoxylin. Magnification: A, B1, C1, and D, x200; B2 and C2, x600.

Detection of Enteroviral Genomic RNA in Myocardial Samples From Patients
The viral genomic RNA was detected in 6 of 11 cases of myocarditis (54.5%) and 3 of 8 cases of DCM (37.5%) but not in any of the 11 control samples. RT-PCR for the ß-actin mRNA was positive in all samples, except for the 3 tissues fixed in Bouin’s solution, which is known to degrade RNA/DNA.⁹ These 3 samples (samples 7, 9, and 15), however, were positive for VP1. The agreement between viral RNA and VP1 detection was statistically significant (=0.6±0.21; the 3 samples were excluded from the test).

	Discussion

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Enteroviral capsid protein VP1 was found in myocardium from patients with myocarditis or DCM. Most of the patients with acute disease died within 1 week after onset in outbreaks of, or sporadic viral myocarditis, when active replication of virus in the heart was expected. Earlier studies reported that 12 of 20 autopsy heart samples from fatal neonatal myocarditis were positive for enteroviral antigen,¹⁰ and CVB3 antigen was detected in samples from 3 pediatric patients with acute idiopathic DCM.¹¹ It is more surprising to find viral antigens in chronic myocardial disease, and this is the first report of detection of VP1 in a high proportion of DCM cases. These results are attributed to patient selection, high sensitivity of the improved technique, and the specificity of mAb 5-D8/1. This antibody has no detectable immunoreactivity with cardiac tissues or with other viruses tested thus far.

The RT-NPCR for detection of enteroviral genomic RNA correlated with the antigen detection. The lower positive rate by the RT-NPCR results from the use of Bouin’s solution. Other factors may include the small size of the tissues and actual quality of the RNA extracts. Unlike RT-NPCR, immunohistochemistry localizes virus. VP1-positive myocytes adjacent to dying or dead myocytes or ongoing inflammatory lesions indicate that enterovirus infection leads directly to the pathological changes in the myocardium.

Previous studies have demonstrated the persistence of enterovirus-specific IgM and enterovirus genomic or negative-stranded RNA in myocardial disease,^{12 13} suggesting the possibility of synthesis of viral proteins in the host cells during virus persistence. In the present study, detection of both viral capsid protein and genomic RNA has provided direct evidence to show that during persistent infection in the heart, enterovirus may produce both viral proteins and RNA or a limited number of progeny, but more likely, defective virus particles, which may facilitate the progression of viral myocarditis to DCM. A recent study indicates that persistence of protease 2A of CVB3 may impair heart functions by cleaving host dystrophin proteins.¹⁴

	Acknowledgments

 
This study was supported by the Ministry of Public Health of China (95-397), Region Rhone-ALPS Convention Program Shanghai (L 087980003), the Wellcome Trust (052954Z97), and the British Heart Foundation (RG/94003).

Received August 3, 1999; revision received November 2, 1999; accepted November 4, 1999.

	References

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