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(Circulation. 1999;99:889-895.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

Enteroviral RNA Replication in the Myocardium of Patients With Left Ventricular Dysfunction and Clinically Suspected Myocarditis

Matthias Pauschinger, MD; Andrea Doerner, PhD; Uwe Kuehl, PhD; Peter L. Schwimmbeck, MD; Wolfgang Poller, MD; Reinhard Kandolf, MD; Heinz-Peter Schultheiss, MD

From Medical Clinic II, University Hospital Benjamin Franklin, Freie Universität Berlin (M.P., A.D., U.K., P.L.S., W.P., H.-P.S.); and the Department of Molecular Pathology, Institute for Pathology, Eberhard-Karls-Universität (R.K.), Tübingen, Germany.

Correspondence to Matthias Pauschinger, MD, Department of Cardiology, University Hospital Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, D-12200 Berlin, FRG.

	Abstract

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Background—Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome.

Methods and Results—We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand–positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26).

Conclusions—These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy.

Key Words: myocarditis • molecular biology • polymerase chain reaction • RNA • viruses

	Introduction

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In many cases of myocarditis (MC), enteroviruses, especially group B coxsackieviruses, are the pathological agents.^{1 2} The whole enterovirus genome is contained within a single-strand monocistronic RNA of positive polarity (plus-strand). During viral replication, this plus-strand RNA first serves as a template for the transcription of a replicate minus-strand RNA intermediate by the RNA-dependent RNA polymerase (3D^pol). This minus-strand RNA is then used as a template again for the 3D^pol to generate multiple copies of viral plus-strand genomes that are translated into enteroviral structural proteins and ultimately packaged into new virions. Therefore, the detection of minus-strand enteroviral RNA is an indicator of the first step of active viral replication taking place.³

The development of highly sensitive molecular biological methods such as in situ hybridization and reverse-transcriptase polymerase chain reaction (RT-PCR) has made possible enteroviral RNA detection in human endomyocardial biopsies.^{4 5 6 7} None of the previous clinical studies, however, addressed the problem of differentiation between active viral replication and latent persistence of enteroviral genomes, which could have important consequences for the clinical course of the disease. This could be one possible explanation for the exactly opposite conclusions drawn by 2 recent studies on the prognostic significance of the presence of enteroviral RNA in patients with MC or dilated cardiomyopathy (DCM). One of these prospective studies⁶ came to the conclusion that the presence of enteroviral RNA in endomyocardial biopsies is a positive prognostic factor with respect to hemodynamic course and survival without the need for heart transplantation. In contrast, the other prospective study⁷ concluded from their data that detection of enteroviral RNA in the myocardium of MC or DCM patients is associated with an adverse prognosis and that the presence of enteroviral RNA is an independent predictor of unfavorable clinical outcome. This contradiction could be explained by different biological activities of the viral infection in the 2 patient collectives. To characterize the activity of the enteroviral infection in more detail, we developed an assay based on RT-PCR and Southern blot hybridization and conducted plus-strand–specific versus minus-strand–specific enteroviral RNA detection. Extensive controls were performed to definitely exclude the possibility of false-positive minus-strand enteroviral RNA detection, as observed in a previous report.⁸

	Methods

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Patients
Forty-five patients with left ventricular dysfunction (defined either as an ejection fraction [EF] 55% or an overall normal EF with regional wall motion disturbance in at least 2 wall segments as analyzed independently by 2 different observers) and clinically suspected MC were enrolled in the study. The diagnosis of MC was suspected on the basis of a typical history with recently occurring cardiac arrhythmia; electrocardiographic changes; reduced exercise tolerance; or atypical chest pain (detailed clinical data are listed in Table 1 and Table 3). Echocardiography, right and left heart catheterization including standard hemodynamic measurements, coronary angiography, and left ventricular angiograms were carried out in all patients⁹ in all of whom coronary, hypertensive, and valvular heart disease as well as restrictive or constrictive heart disease were excluded.

View this table: [in this window] [in a new window]   Table 1. Clinical, Hemodynamic, Histological, and Immunohistological Characteristics of Enrolled Patients

View this table: [in this window] [in a new window]   Table 3. List Patients, Including Demographic, Hemodynamic, Histological, Immunohistological, and Molecular-biological Characteristics

Endomyocardial Biopsies
At least 5 endomyocardial right ventricular biopsies were taken from each patient. One or two biopsies were submitted to molecular biological analysis for enteroviral RNA. The other biopsies were analyzed histologically and immunohistologically to assess myocardial inflammation. The analyses were performed in blinded fashion by persons unaware of patient data and history. Biopsies from explanted hearts of 26 patients having undergone heart transplantation (coronary heart disease [n=24] and valvular disease [n=2]) served as negative controls. These biopsies obtained at the time of heart transplantation were immediately frozen in liquid nitrogen in the operating room to preserve messenger and enteroviral RNA, respectively.

All procedures were performed in accordance with ethical standards and with the Helsinki Declaration of 1975. All patients gave informed consent for all of the invasive studies performed.

Histology and Immunohistology
Hematoxylin-eosin staining of paraffin sections was carried out and analyzed according to standard methods.¹⁰ In addition, cryostat sections were analyzed for infiltrating CD3 T-lymphocytes as published previously.¹¹

RT-PCR and Southern Blot Analysis
All patients were included in the study prospectively before completion of RT-PCR. One hundred nanograms total RNA¹² of each biopsy was transcribed into cDNA by MMLV reverse transcriptase with random primers according to the supplier's recommendations (BRL). Identical reactions containing no RNA template served as negative controls and were consistently negative in all further analyses. In separate experiments to determine the polarity of the RNA, the cDNA of plus-strand enteroviral RNA was synthesized in a 20-µL solution containing 100 ng total RNA, 0.5 mmol/L deoxynucleotide triphosphate, 0.06 pmol 3' coxsackie primer, and 200 IU MMLV reverse transcriptase as described by the manufacturer. The cDNA of minus-strand enteroviral RNA was synthesized in the same way using 0.06 pmol 5' coxsackie primer instead of 3' coxsackie primer (Table 2). The cDNA thus obtained was heated to 95°C for 30 minutes to inactivate the reverse transcriptase activity and then incubated at 0°C for 10 minutes (Figure 1). Each sample was directly used in the RT-PCR with a 35-cycle program consisting of denaturation at 94°C for 45 seconds, annealing at 61°C for 45 seconds, and extension at 72°C for 90 seconds in a standard PCR buffer (1.5 mmol/L MgCl₂, 50 mmol/L KCl, 10 mmol/L TRIS-HCl, pH 8.3, 0.01% gelatin, 0.3 µmol/L primers, 0.2 mmol/L dNTPs, 2.5 U Taq-polymerase).¹³ The oligonucleotide sequences chosen for enteroviral RNA amplification are located in the consensus enteroviral sequences of the noncoding 5' region and therefore present a high degree of group specificity (Table 2) and detect different types of enteroviruses (coxsackievirus B1-B6, coxsackievirus A9, poliovirus 1, ECHO-viruses 11 and 12).^{3 14} The amplified RT-PCR product has a length of 314 bp. A cloned cDNA of coxsackievirus B3 served as a positive control.¹⁵ For further positive controls, coamplification of endogenous ß-actin and enteroviral RNA was carried out with specific ß-actin primers (Table 2).¹⁶ The RT-PCR products were separated by gel electrophoresis and stained with ethidium bromide in standard methods. The ß-actin bands were then visualized by ultraviolet light (254 nm) with a length of 461 bp (molecular weight marker pBR328 DNABglI+pBR328 DNAHinfI) (Figure 2). This was followed by Southern blot hybridization with a 5' P³²-labeled oligonucleotide (cox hyb: 5'-CGAAGTAGTTGG-CCGGATAAC-3') in standard methods^{3 14} (membranes were washed twice in 6xSSC and 0.1% SDS at room temperature and in 0.1x SSC and 0.1% SDS for 30 minutes at 50°C) (Figure 2).

View this table: [in this window] [in a new window]   Table 2. Primers Used for Detection of Enteroviral RNA by RT-PCR

View larger version (78K): [in this window] [in a new window]   Figure 1. Need of heat inactivation of the reverse transcriptase in a strand-specific RNA detection assay is shown. Enteroviral minus-strand RNA transcribed in vitro from the T7 promotor of the cloned enterovirus cDNA, used in lanes 1 to 9 as a template, was specifically reverse transcribed using 5' coxsackie primer (minus-strand specific detection) in lines 1 and 9. No signal should be detected when using the 3' coxsackie primer (plus-strand specific detection) for reverse transcriptase in lanes 2 to 8. Complete elimination of false-positive signals seen in these lanes, however, was only possible by using a rigorous heat inactivation protocol. Time course of reverse transcriptase inactivation caused by boiling is shown in an ethidium bromide–stained agarose gel (top) and after Southern hybridization with a P32-labeled enterovirus-specific probe (cox hyb) (bottom) in lanes 2 to 8 (heat inactivation time 5 minutes [lane 2], 10 minutes [lane 3], 15 minutes [lane 4], 20 minutes [lane 5], 30 minutes [lane 6], 45 minutes [lane 7], and 60 minutes [lane 8]). M indicates molecular weight marker.

View larger version (100K): [in this window] [in a new window]   Figure 2. Strand-specific enteroviral RNA detection in endomyocardial biopsies from 3 different patients. Top, Ethidium bromide–stained agarose gel; bottom, corresponding and after Southern hybridization with an enterovirus-specific P32-labeled probe (cox hyb). In lanes 1, 4, and 7 (RT-PCR with random primer for RT and ß-actin plus coxsackie primers for subsequent PCR), 2 bands are visible on the agarose gel, corresponding to the amplified ß-actin (461 bp) and/or enteroviral cDNA (314 bp). After Southern blot hybridization with the P32-labeled cox hyb probe, only 1 signal is still detectable that corresponds to the amplified enteroviral cDNA. In lanes 2, 5, and 8, RT-PCR was performed with 3'coxsackie primers for RT and ß-actin plus coxsackie primers for subsequent PCR. The signals seen in lanes 2 and 5 correspond to enteroviral plus-strand cDNA. In lanes 3, 6, and 9, RT-PCR was done with 5'coxsackie primers for RT and ß-actin plus coxsackie primers for subsequent PCR. The signal seen in lane 3 corresponds to enteroviral minus-strand cDNA. In summary, patient A (lanes 1 to 3) displays active enteroviral replication including minus-strand detection; patient B (lanes 4 to 6) shows latent virus persistence (no minus strand detectable); patient C (lanes 7 to 9) is enterovirus negative. M indicates molecular weight marker; C, positive control of coxsackievirus B3 cloned DNA [314 bp]).

Strand-Specific Enteroviral RNA Detection
Throughout the detection procedure for minus-strand enteroviral RNA, complete inactivation of the reverse transcriptase after cDNA synthesis was essential for strand specificity. Therefore, the specificity of the strand-specific enteroviral RNA detection assay was tested by using T7 and SP6, respectively, in in vitro synthesized sense and antisense transcripts of coxsackie B3 enteroviral RNA from the 5' noncoding region (577 bp). As a control for inactivation of the reverse transcriptase, enteroviral RNA transcripts were incubated with the 3' coxsackie primer in the reverse transcription mixture (Figure 1). This reverse transcription mixture was boiled for 0 to 60 minutes to inactivate the enzyme activity of the reverse transcriptase. After the enzyme heat inactivation, this mixture was amplified by 35 cycles of RT-PCR with the 3' and 5' coxsackie primers, respectively. Positive signals for minus-strand RNA were completely absent after boiling for more than 20 minutes in all cases. The positive signal for minus-strand RNA until 15 minutes of boiling appears to be the result of residual reverse transcriptase activity in the RT-PCR reaction mix. Therefore, all samples analyzed in the strand-specific detection assay were boiled for 30 minutes after first-strand cDNA synthesis to definitely avoid false-positive results (Figure 3).

View larger version (71K): [in this window] [in a new window]   Figure 3. Specificity is documented of the assay used in this study to unequivocally distinguish plus-strand from minus-strand enteroviral RNA. Shown is the analysis of in vitro transcribed enteroviral RNA of positive polarity and negative polarity, respectively. Top, Ethidium bromide–stained agarose gel; bottom, after Southern blot hybridization with an enterovirus-specific P32-labeled probe (cox hyb). Lanes 1 and 2 used in vitro synthesized plus-strand RNA reverse transcribed with 3' (lane 1) and 5' (lane 2) coxsackie primers, respectively. Signal seen in lane 1 but not in lane 2 corresponds to the specific detection of enteroviral plus strand after PCR. Lanes 3 and 4 used in vitro synthesized minus-strand RNA reverse transcribed with 3' (lane 3) and 5' (lane 4) coxsackie primers, respectively. Signal seen in lane 4 but not in lane 3 corresponds to the specific detection of enteroviral minus strand after PCR. Lane 5, Negative control RT-PCR of minus-strand enteroviral RNA without RT; lane 6, negative control of RT-PCR without template. M indicates molecular weight marker; C, positive control of coxsackievirus B3 (314 bp).

Statistical Analysis
All values are expressed as mean±SD.

	Results

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Patients and Hemodynamic Data
Forty-five patients (age 48±14 years, range 22 to 75 years) with clinically suspected diagnosis of MC were enrolled in the study. All patients had regional or global left ventricular dysfunction as assessed by ventriculography and echocardiography; mean EF was 47%±13%. In addition, the echocardiographic data showed an enlarged mean left ventricular end-diastolic diameter (LVEDD) (62±10 mm). The detailed clinical and hemodynamic data as well as the indications for taking endomyocardial biopsies are listed in Table 1 and Table 3.

Histology and Immunohistology
The histological investigation of the endomyocardial biopsies resulted in the diagnosis of borderline MC in 6 (13%) of the studied patients. Immunohistological analysis of the endomyocardial biopsies showed increased mean CD3 T-lymphocytes (3.3±6.9 T-lymphocytes/HPF) (Table 1).

Strand-Specific Enteroviral RNA Detection
RT-PCR combined with Southern blot hybridization was used to examine the myocardial biopsies of all 45 patients with left ventricular dysfunction and clinically suspected MC for plus-strand and minus-strand enteroviral RNA. At the same time, 26 samples from the control population were examined. In all 71 amplifications, the internal ß-actin–positive control was visible in the ethidium-bromide–stained agarose gel under ultraviolet light (Figure 2). This excludes false-negative biopsy results caused by degradation of extracted RNA. In patients with the left ventricular dysfunction and clinically suspected diagnosis of MC, plus-strand enteroviral RNA was detected in 18 (40%) cases. In ten (56%) of these 18 plus-strand–positive patients, detection of minus-strand enteroviral RNA was possible. These data demonstrate that a significant fraction of patients (22%) with left ventricular dysfunction and clinically suspected MC had active enteroviral RNA replication in the myocardium.

In contrast, all 26 patients from a control group were negative when myocardial tissue was examined for enteroviral RNA by RT-PCR.

	Discussion

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Transcription of minus-strand RNA from the plus-strand enteroviral genomic template is the essential first step of enteroviral replication. Therefore, selective RT-PCR detection of the minus-strand RNA is an indicator of active enteroviral RNA replication of enteroviral genomes.³ In our study, strand-specific enteroviral RNA detection was performed in human endomyocardial biopsies from 45 patients with left ventricular dysfunction and clinically suspected MC. Eighteen (40%) of 45 of the patients studied were positive for plus-strand enteroviral RNA in the myocardium. Ten (56%) of these 18 patients also had minus-strand enteroviral RNA, indicating that a significant fraction of patients with left ventricular dysfunction and clinically suspected MC had active enteroviral RNA replication in the myocardium.

After introduction of highly sensitive molecular biological methods in the diagnosis of viral heart disease, the detection of enteroviral RNA in human endomyocardial biopsies became possible.^{4 5 6 7} However, the clinical relevance of enteroviral RNA detection in endomyocardial biopsies of patients with DCM or MC is highly controversial, as discussed in 2 recently published reports.^{6 7} The hemodynamic status of the enrolled patients in both studies was comparable with respect to EF and LVEDD. However, despite the comparable degree of left ventricular dysfunction in both studies as a well known parameter of prognosis in chronic heart failure,^{17 18} the clinical course of the analyzed patients in both studies was the opposite. Nevertheless, 1 study⁶ concluded that the presence of enteroviral RNA is a favorable prognostic factor, whereas the other study⁷ found it to be associated with an adverse outcome. These controversial results of the 2 studies, despite comparable hemodynamic impairment of left ventricular dysfunction, might possibly be explained by different biological activities of viral heart disease.

On the basis of our knowledge on enterovirus biology, an assay distinguishing latent persistence and active replication could possibly resolve this problem. Whether or not this differentiation is sufficient to completely resolve the issue, this kind of more thorough analysis should be incorporated in future prospective studies of MC. Moreover, distinction of latent persistence versus active enteroviral replication in the myocardium might also improve our understanding of this apparently highly dynamic human viral disease. Considering the controversial results of several small therapy trials of human MC,^{6 19 20 21} it seems appropriate to characterize as exactly as possible the disease state of patients to be assigned to one or another treatment regime. According to the Dallas¹⁰ criteria, 13% of the biopsies of our patients with left ventricular dysfunction and clinically suspected MC were diagnosed as having borderline MC. This incidence of histologically positive results in patients with clinically suspected MC is comparable with the Myocarditis Treatment Trial.¹⁹ In this trial, active plus borderline MC was diagnosed histologically by the pathologists at the participating centers in 10% of patients with clinically suspected MC. However, a later review by an independent pathology panel of 107 enrolled endomyocardial biopsies came to the conclusion that only 64% actually met the Dallas criteria for active MC or borderline MC.^{10 19} This emphasizes the considerable difficulties associated with the histological diagnosis of MC. In part, the low incidence of histologically positive results in patients with clinically suspected MC¹⁹ may be due to the fact that MC is often focal and therefore missed by the standard biopsy procedure. In addition, cellular infiltrates are sparse, and by using standard histological techniques it is difficult to distinguish between noninflammatory cells (for example, fibroblasts or pericytes) and infiltrating lymphocytes.²² For this reason, we additionally used immunohistological techniques to identify low-level lymphocytic infiltrates in myocardial biopsies.¹¹ Although 87% of our endomyocardial biopsies classified as negative according to the Dallas criteria showed inflammatory cell infiltrates, immunohistology did not help to distinguish between patients with and those without enteroviral replicative intermediates.

False-positive minus-strand detection caused by reverse transcriptase activity during RT-PCR was ruled out in our study by exhaustive inactivation of the enzyme by boiling for 30 minutes at the end of the reverse transcription. In addition, strand specificity was documented by analyzing in vitro transcribed cloned enteroviral cRNA of positive polarity and negative polarity, respectively, as templates. The plus strand was transcribed from the SP6 promotor and the minus strand from the T7 promotor of our enteroviral cDNA clone. Former problems⁸ with strand specificity were thus eliminated. Furthermore, our assay system unequivocally differentiated between virus-positive (strong signal) and virus-negative patients (no signal). Intermediate results as previously reported for the slot blot assay described by Why et al⁷ with a defined cut-off value of the hybridization index were never observed in any of our patients.

Summary
Our results show that a significant fraction of patients (22%) with left ventricular dysfunction and clinically suspected MC had active enteroviral RNA replication in their myocardium. Therefore, molecular genetic differentiation between active viral replication and latent viral persistence should be considered in the design of prospective clinical studies on the clinical course of this apparently dynamic viral disease. This differentiation should be particularly important in future studies evaluating different therapeutic strategies.

Received July 31, 1998; revision received November 4, 1998; accepted November 11, 1998.

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