**4.2 The chronic persistent viral Infection in heart tissues**

#### **4.2.1 The enterovirus model**

After the acute myocarditis phase, persistence can be observed as described during human cardiac enterovirus (EV) infection. In some previous published studies, detection of viral genome has been demonstrated in patients with myocarditis and in patients with DCM, but it is unusual that replication competent virus can be isolated from the myocardium in patients with myocarditis (Andréoletti et al., 2009; Chapman & Kim, 2008a; Copper, 2009). The concept that EV endomyocardial persistent infections are the etiological cause of a subset of idiopathic DCM cases is supported by the detection of enterovirus genomic sequences and enteroviral capsid protein VP1 in up to 35 % of explanted heart tissues from end-stage DCM patients (Andreoletti et al. 2009; Li et al., 2000). In a previous published study, we observed that enteroviruses can persist with or without active viral replication in cardiac tissue of patients with end-stage dilated cardiomyopathy (Andréoletti et al., 2000). Enterovirus genome was detected in 25 of 70 patients with IDCM and, of these patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 22 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects (Andréoletti et al., 2000). These findings demonstrated that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium. Moreover, we demonstrated that enteroviral capsid protein VP1 was present in myocardial tissues from some patients with dilated cardiomyopathy and suggested that the pattern of VP1 detection may correlate with disease stage and severity. These data suggested that viral protein synthesis might be involved in persistent enterovirus infection in the pathogenesis of DCM (Li et al., 2000). It was observed that the ratio of positive- to negative-strand enteroviral RNA was greater with active virus replication than with persistent virus infection where a viral capsid protein synthesis activity was also evidenced in heart cardiac tissues (Andréoletti et al., 2000; Li et al., 2000, Chapman & Kim, 2008a). This slow viral replication could be explained by the existence of 5'NC genomic deleted viral forms that could be related to the development of slow replicating viral forms in heart tissues (Chapman et al., 2008b). As persistent expression of CVB proteins and 2Aprotease (2Apro) alone are sufficient for induction of cardiomyopathy in the mouse and as detection of HEV TD genomes in adult human heart disease is likely due to persistent HEV TD genomes, there is now a new hypothetical mechanism to link of acute viral myocarditis with postviral DCM (Chapman et al. 2008b).

#### **4.2.2 The Parvovirus B19 model**

Concerning the Parvovirus B19 virus, a persistent infection was detected in intra-cardiac endothelial cells of small arterioles and veins of patients with chronic cardiomyopathies by in situ hybridization and PCR techniques. This persistent PVB19 infection associated with a low viral replication may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells and secondary myocyte necrosis. (Duechting et al., 2008). The molecular mechanisms responsible for the reactivation

al., 2009). During this active phase of replication, the development of the classical clinical signs of myocarditis is usually observed (Andréoletti et al., 2007; Magnani & Dec, 2006).

After the acute myocarditis phase, persistence can be observed as described during human cardiac enterovirus (EV) infection. In some previous published studies, detection of viral genome has been demonstrated in patients with myocarditis and in patients with DCM, but it is unusual that replication competent virus can be isolated from the myocardium in patients with myocarditis (Andréoletti et al., 2009; Chapman & Kim, 2008a; Copper, 2009). The concept that EV endomyocardial persistent infections are the etiological cause of a subset of idiopathic DCM cases is supported by the detection of enterovirus genomic sequences and enteroviral capsid protein VP1 in up to 35 % of explanted heart tissues from end-stage DCM patients (Andreoletti et al. 2009; Li et al., 2000). In a previous published study, we observed that enteroviruses can persist with or without active viral replication in cardiac tissue of patients with end-stage dilated cardiomyopathy (Andréoletti et al., 2000). Enterovirus genome was detected in 25 of 70 patients with IDCM and, of these patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 22 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects (Andréoletti et al., 2000). These findings demonstrated that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium. Moreover, we demonstrated that enteroviral capsid protein VP1 was present in myocardial tissues from some patients with dilated cardiomyopathy and suggested that the pattern of VP1 detection may correlate with disease stage and severity. These data suggested that viral protein synthesis might be involved in persistent enterovirus infection in the pathogenesis of DCM (Li et al., 2000). It was observed that the ratio of positive- to negative-strand enteroviral RNA was greater with active virus replication than with persistent virus infection where a viral capsid protein synthesis activity was also evidenced in heart cardiac tissues (Andréoletti et al., 2000; Li et al., 2000, Chapman & Kim, 2008a). This slow viral replication could be explained by the existence of 5'NC genomic deleted viral forms that could be related to the development of slow replicating viral forms in heart tissues (Chapman et al., 2008b). As persistent expression of CVB proteins and 2Aprotease (2Apro) alone are sufficient for induction of cardiomyopathy in the mouse and as detection of HEV TD genomes in adult human heart disease is likely due to persistent HEV TD genomes, there is now a new hypothetical mechanism to link of acute viral myocarditis with postviral DCM (Chapman et al. 2008b).

Concerning the Parvovirus B19 virus, a persistent infection was detected in intra-cardiac endothelial cells of small arterioles and veins of patients with chronic cardiomyopathies by in situ hybridization and PCR techniques. This persistent PVB19 infection associated with a low viral replication may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells and secondary myocyte necrosis. (Duechting et al., 2008). The molecular mechanisms responsible for the reactivation

**4.2 The chronic persistent viral Infection in heart tissues** 

**4.2.1 The enterovirus model** 

**4.2.2 The Parvovirus B19 model** 

of latent parvovirus B19 infection, the influence of immune activation triggering parvovirus B19 replication and chronic myocarditis, and immune-independent viral pathogenesis remains to be assessed (Bock et al., 2010).

#### **4.3 The chronic latent viral cardiac infections**

In cases of *herpesviruses* (HSV1, HSV-2 , HCMV, HHV-6) cardiac infection a latent phase can occur subsequently to the acute phase of infection as demonstrated in a mice model (Grodums & Zbitnew, 1976). During this HSV experimental latent infection, HSV-DNA can be identified as agents of a persistent heart infection in cardiomyocytes, fibroblasts or Schwann cells, which has be seen in unmyelitinated axons in murine heart tissues. In human subjects, CMVH was detected in cardiomyocytes and in cardiac fibroblasts of patients with histological proven myocarditis (Schönian et al., 1995). Moreover, it was not possible to detect viral mRNA coding for structural proteins known as late proteins but only mRNA coding for viral enzymatic proteins (early proteins) related to the regulation of viral replication or associated with the HCMV DNA replication (Lenzo et al., 2002) Whatever, the cellular sites as well as the mechanisms of latency and reactivation of the *herpesviruses* (EBV, HCMV and HHV6) in human heart tissues remain to be assessed (Andréoletti et al., 2009; Cooper, 2009; Dennert et al., 2008).

#### **5. Viral causes for human acute or chronic myocarditis**

Human Enteroviruses, (*picornaviridae*), specifically Coxsackie group B serotypes, Parvovirus B19, HHV6 of the B type and the adenovirus are the most frequently etiological viral agents implicated in the acute myocarditis of the child or the young adult (<35 year-old) (Andréoletti et al., 2009; Bowles et al., 2003; Feldman & McNamara, 2000; Kühl et al., 2005b; Magnani & Dec, 2006) (Table 2). Moreover HHV-1, Adenovirus, myxoviruses and also paramyxoviruses including respiratory syncytial virus (RSV), *influenza* and *parainfluenza*  strains can also induce an acute infection of cardiac tissue as previously demonstrated in reported acute myocarditis cases developed in immunocompetent patients (Bowles et al., 2003; Dennert et al., 2008). Other viruses as EBV or CMV are also associated with this pathology after heart transplantation. HIV or HCV can be also etiological agents of myocarditis (Matsumori et al., 2006; Sudano et al., 2006) (Table 2).

Recent data showed that it was possible to detect viruses in 71% of the cases of acute myocarditis using molecular techniques for the virological analysis of cardiac biopsy samples (Table 2) (Kühl et al., 2003). Co-infections were found in more than 12% of the cases, generally associating HHV6 and Parvovirus B19. HHV6 seems to be an important cofactor of myocarditis due to Parvovirus B19. HHV6 may enhance the pathogenicity of Parvovirus B19 through alterations of the extracellular matrix and modulation of the expression levels of the PVB19) receptor (P-antigen) on endothelial cells facilitating infections of the coronary vascular endothelium (Table 2) (Kühl et al., 2005a).

Concerning chronic myocarditis, there is no clinical data from transverse or longitudinal studies indicating the incidence of various viral causes of cardiac infection. However, viral persistence in the myocardium was associated with ventricular dysfunction whereas viral genome clearance was related to the hemodynamic improvement (Kühl et al. 2005a, 2005b). As in the cases of acute myocarditis, recent studies showed the interest to test a broad panel of cardiotropic viruses at the DCM stage. Thus, in a case series of 245 patients with clinically suggested DCM, one or more viruses were detected in 67% of the cases (Table 2) (Kühl et al.,

al., 2004).

**6.1 Endomyocardial biopsy (EMB)** 

histological lesions (Baughman, 2006).

**6.2 Histopathological evaluation of cardiac biopsy samples** 

(Yilmaz et al., 2010).

Viral Myocarditis: Physiopathology and Diagnosis 95

of viral serological assay remains of a limited interest in clinical practice (Mhafoud et al., 2011). A recent European study demonstrated that virus serology has no relevance for the diagnosis of myocardial infection in young adults; this study indicated that comparatively to the molecular analysis of endomyocardial biopsy tissues, the positive predictive value was 25% and that the negative predictive value was 49% (Mhafoud et al., 2011).Therefore the etiological diagnosis of viral myocarditis is based on the detection of the viruses or viral components (proteins or genomes) in peripheral blood samples at the time of viremia phase (clinically characterized by fever), but also at the entry and the excretion sites (throat, urine and stool samples), and in heart tissue samples that corresponds to the organ site of viral replication. Therefore, endomyocardial biopsy remains the gold standard for unequivocally establishing the histopathological and virological diagnosis of unexplained cardiomyopathies as acute or chronic myocarditis (Li et al., 2000; Mahrholdt et al., 2004). Its clinical impact on prognosis and treatment largely depends on establishing a rapid and standardized set of diagnostic methods including histopathological and virological analyses of endomyocardial tissue taken by endomyocardial biopsy (Aretz et al., 1987; Mahrholdt et

As recommended by the Heart Failure Society of America and the Heart Failure Association of the European Society of Cardiology, the implementation of a right or left ventricular EMB is indicated in the case of acute symptoms of heart failure refractory to standard management, a substantial worsening of ejection fraction despite optimized pharmacological therapy, the development of hemodynamically significant arrhythmias, an heart failure with concurrent rash, fever, or peripheral eosinophilia, an history of collagen vascular disease such as systemic lupus, erythematosus, scleroderma, or polyarteritis nodosum, and a new-onset cardiomyopathy in the presence of known amyloidosis, sarcoidosis, or hemochromatosis when no obvious cause, in particular ischemic, could be established (Mahrholdt et al., 2004). EMB remains the gold standard for unequivocally establishing the diagnosis of unexplained cardiomyopathy Li et al., 2000). However, its sensitivity and its specificity are limited by the often-focal distribution of the specific

Cardiac biopsy samples should be obtained in more than one area of the right ventricular septum and the number of samples should range from 5 to 10 of a volume from 1 to 2 mm3. Five of these samples should be fixed in neutralized 10% formalin or 10% PFA; five should be flash-frozen or placed immediately conserved at –80°C to perform further classical or molecular virological techniques (Mahrholdt et al., 2004). Interestingly, it has been recently demonstrated that there were no differences in the number of positive left ventricular-EMB, right ventricular-EMB, or left ventricular- and right ventricular-EMB findings when related to the site of cardiovascular magnetic resonance– based late gadolinium enhancement. Preferential biopsy in regions showing late gadolinium enhancement on cardiovascular magnetic resonance does not increase the number of positive diagnoses of myocarditis.

Histological evaluation of cardiac biopsies from patients with clinically suspected myocarditis is routinely done according to the Dallas criteria (Cooper, 2009). Since


*(-) Not determined; \* Prevalence determined in cases of solid organ transplantation.* 

Table. 1. Prevalence of viruses detected by molecular biology-based techniques in cardiac tissue samples taken from patients with acute myocarditis or dilated cardiomyopathy.

2005a). Moreover in these patients, the absence of associated myocardial inflammation suggests that viral persistence can be responsible for a modulation of the immune response, which would be decreased from the beginning of the chronic phase of myocarditis (Kawai, 1999; Yajima & Knowlton, 2009).
