Introduction

Although the course of viral myocarditis in most patients is subclinical, some patients develop a severe, sometimes fatal disease.1'2 In susceptible individuals, viral myocarditis may result in sudden death or the patient may present with cardiac failure with a clinical picture similar to that of idiopathic dilated cardiomyopathy (DCM).3 Enteroviruses, and especially coxsackieviruses of group B (CVB), are the most common infectious agents.2,4

Enteroviruses of the Picornaviridae are nonenveloped icosahedral viruses that contain a single plus-strand RNA genome of about 7,500 bp. The genomic viral RNA, which is polyadenylylated at its 3'-end, serves as messenger for the translation of virus-directed structural and nonstructural proteins and as a template for transcription of a minus-strand

RNA intermediate by the virus-encoded RNA-dependent RNA-polymerase 3Dpo1. Minusstrand RNA is transcribed again by 3Dpol, providing large amounts of plus-strand RNA genomes, which are encapsidated into new virions. The acute infectious cycle is usually completed by cell lysis and release of newly synthesized virus particles.

Other viruses, such as adenoviruses,5-9 cytomegalovirus,5,6,9-11 herpes simplex virus,5,6

Epstein-Barr virus,12 human herpesvirus 6,13 human immunodeficiency virus (HIV),14 parvovirus B19 (PVB19),15-17 influenza A,18 mumps virus,19 rubella virus,20 and hepatitis

C virus,21,22 have also been linked with acute and chronic myocarditis. However, few data are available to establish a direct role for these viruses in the pathogenesis of the disease.

Diagnosis of viral heart disease is difficult and generally depends on clinical criteria combined with histologic findings in endomyocardial biopsy specimens. Morphologic features in heart muscle of patients thought to have viral myocarditis, however, cannot be definitely distinguished from inflammatory heart diseases of nonviral origin. Therefore, virologic diagnosis may be of great value in differentiating heart disease of viral etiology from that of other causes. For a long time, diagnosis of myocardial virus infections relied on the time-consuming virus isolation in tissue culture or on serologic tests.23 However, attempts to isolate virus were unrewarding because patients are rarely viremic or excrete infectious virus in stool or nasopharyngeal secretions beyond the acute phase of virus infection. In addition, the conventional virologic techniques, including virus isolation and immunohistochemical staining of serotype-specific viral antigens, were successful only in cases of fulminant acute viral myocarditis owing to the low sensitivity of these methods.24

In the last decade, advances in molecular biology resulted in the development of more sensitive assays for the detection of viral genomes in the myocardium. The establishment of an in situ hybridization technique for the visualization of enteroviral genomes at the cellular level allowed definition of the role of enteroviruses in the induction and maintenance of myocarditis as well as study of the molecular basis of pathogenicity. By application of enterovirus group-specific in situ hybridization, it was demonstrated that enterovirus infection is detectable in a significant proportion of patients with acute and chronic myocarditis and also in patients with end-stage dilated cardiomyopathy, indicating that chronic myocardial injury may be associated with persistent enterovirus infection.25-28 The capability of enteroviruses to persist in myocardial cells was confirmed by in situ hybridization studies in various murine models of chronic myocarditis, demonstrating that CVB3, typically a cytolytic virus, is capable of evading the immunologic surveillance in a host-dependent manner.29-32 In addition to in situ hybridization, the application of the polymerase chain reaction (PCR) has offered new possibilities for sensitive and rapid detection of infectious agents in viral heart disease. Primer pairs and amplification protocols have been published for the identification of RNA viruses (eg, enteroviruses) and also for DNA viruses (eg, Epstein-Barr virus and adenoviruses) in frozen as well as in paraffin-embedded heart muscle tissue.4-10'12-22 This chapter focuses on the currently available methods for detecting viral nucleic acids in the myocardium with special reference to the most common cause of viral heart disease—the enteroviruses. The technical principles discussed in this review, which are of general importance for the diagnosis of virus infections, comprise detection of viral nucleic acid sequences by blot hybridization, light and electron microscopic in situ hybridization, PCR, and in situ PCR.

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