**1. Introduction**

242 Myocarditis

Vossen, M. T., Westerhout, E. M., Soderberg-Naucler, C., and Wiertz, E. J. (2002). Viral

Wang S, L. J., Wang M, Zhang J, Wang Z. (2010). Treatment and prevention of experimental

Wiegand, J. A., Torgersen, C., Bloechlinger, S., Takala, J., and Dunser, M. W. (2010).

Woodruff, J. (1980). Viral myocarditis. *The American Journal of Pathology,* Vol. 101 (November

Wu, C. Y., Feng, Y., Qian, G. C., Wu, J. H., Luo, J., Wang, Y., Chen, G. J., Guo, X. K., and

Zajonc, D. M., Elsliger, M. A., Teyton, L., and Wilson, I. A. (2003). Crystal structure of CD1a

*Molecular Biology,* Vol. 377 (April 2008), No. 4, pp. 1104-16, ISSN 0022-2836. Zheng, Y., He, Y., Deng, J., Lu, Z., Wei, J., Yang, W., Tang, Z., Li, B., Zhang, J., Wang, L.,

Zingoni, A., Sornasse, T., Cocks, B. G., Tanaka, Y., Santoni, A., and Lanier, L. L. (2005). NK

*Immunology,* Vol. 4 (August 2003), No. 8, pp. 808-15, ISSN 1529-2908. Zajonc, D. M., Savage, P. B., Bendelac, A., Wilson, I. A., and Teyton, L. (2008). Crystal

*Immunology,* Vol. 162 (October 2010), No. 1, pp. 178-87, ISSN 0009-9104. Yokoyama, A. (1988). Natural killer cells in dilated cardiomyopathy. *The Tohoku Journal of Experimental Medicine.* Vol. 154 (April 1988), No. 4, pp. 335-44, ISSN 0040-8727. Yuan, W., Dasgupta, A., and Cresswell, P. (2006). Herpes simplex virus evades natural killer

2002), No. 8, pp. 527-42, ISSN 0093-7711.

2010), No. 2, pp. 107-113, ISSN 0008-6312.

1980), No 2, pp. 425-483, ISSN 0002-9440.

7(August 2006), No. 8, pp. 835-42, ISSN 1529-2908.

(February 2005), No. 4, pp. 451-4, ISSN 0161-5890.

ISSN 0043-5325.

ISSN 8755-6863.

immune evasion: a masterpiece of evolution. *Immunogenetics,* Vol. 54 (November

autoimmune myocarditis with CD28 superagonists. *Cardiology,* Vol. 115 (January

Influenza A(H1N1) infection and severe cardiac dysfunction in adults: A case series. *Wiener Klinische Wochenschrift,* Vol. 123(February 2011), No. 3-4, pp. 120-123,

Wang, Z. J. (2010). alpha-Galactosylceramide protects mice from lethal Coxsackievirus B3 infection and subsequent myocarditis. *Clinical and Experimental* 

T cell recognition by suppressing CD1d recycling. *Nature Immunology*, Vol.

in complex with a sulfatide self antigen at a resolution of 2.15 A. *Nature* 

structures of mouse CD1d-iGb3 complex and its cognate Valpha14 T cell receptor suggest a model for dual recognition of foreign and self glycolipids. *Journal of* 

Zhao, H., Li, X., Yu, Z., Song, P., Ma, Y., Li, Y., and Li, C. (2010). Hospitalized children with 2009 influenza a (H1N1) infection in Shenzhen, China, novemberdecember 2009. *Pediatric Pulmonology*. Vol. 46 (March 2011), No. 3, pp. 246-252,

cell regulation of T cell-mediated responses. *Molecular Immunology,* Vol. 42

Myocarditis is a devastating cardiac disease causing death in children and young adults worldwide (Esfandiarei et al. 2008). The disease is clinically characterized by inflammation of the myocardium and degeneration of myocytes. Clinical symptoms of viral myocarditis range from flu-like and/or gastrointestinal illness to ventricular dysfunction ending commonly in heart failure. Acute disease is accompanied by multiorgan abnormalities and presents mostly in neonates and young children. Chronic disease occurs in one third of patients and is likely a consequence of autoimmune-mediated myocardial injury and viral persistence. As cardiac myocytes are destroyed by virus and/or self-induced cytopathic immune effects, excessive repair or fibrosis of myocardial tissue impairs disease progression rather than protects the tissue from further damage. Fibrosis or scar tissue, can lead to abnormal ventricular architecture that inevitably leads to the disruption of its function. At this stage of disease, chronic myocarditis progresses to dilated cardiomyopathy and can eventually lead to congestive heart failure (Esfandiarei et al. 2008).

The exact cause for myocarditis is still unknown though pathogen infections, hypersensitivity reactions, and systemic and autoimmune diseases are all likely contributing factors. It is suggested that acute viral myocarditis that progresses to chronic disease mirrors the clinical pathology observed with dilated cardiomyopathy patients and is likely the result of an inappropriate immune response after virus infection that leads to chronic inflammation and virus persistence (Escher et al. 2011).

Tracking the incidence of myocarditis is challenging. It is difficult to determine the potential disease burden to populations since there is a range in clinical symptoms associated with disease and endomyocardial biopsy to diagnose disease is rarely practised (Blauwet et al. 2010). Though mounting evidence of viral genomes recovered from chronic dilated cardiomyopathy patients provides some insight in to the potential burden of this devastating cardiac disease (Kuhl et al. 2005). Viral-induced myocarditis and dilated cardiomyopathy leads to a worse prognosis than other possible myocarditis/dilated cardiomyopathy etiological agents. Isolation of enteroviral RNA from endocardial biopsies of myocarditis and dilated cardiomyopathy patients renders these patients six times more susceptible to death after two years from diagnosis compared to virus-negative patients (Why et al. 1994). Sex is another contributing factor to disease susceptibility. Initially, it was

The Key Players of Coxsackievirus-Induced Myocarditis 245

Fig. 1. Tentative coxsackievirus life cycle. The general host and virus components suggested

Once the virus enters the cytosol and uncoats, its positive-sense genome is released in the cytosol for translation and later, transcription. As a polyprotein comprised of the virus proteins VP4, VP3, VP2, VP, 2A, 2B, 2C, 3A, 3B, 3C, and 3D emerges from translation at the rough endoplasmic reticulum (ER), it is cleaved into its respective structural and functional proteins. The virally encoded 3Dpol is a RNA-dependent RNA polymerase that transcribes viral positive-sense RNA in to negative-sense RNA strands that serve as intermediates for the transcription of multiple positive-sense RNA strands needed for new progeny virions. After synthesis, the newly generated positive-RNA strands are packaged in new virus particles formed by the newly generated structural and functional virus proteins. The new progeny viruses are then released via plasma membrane by a mechanism likely mediated by viral

to contribute to production of new coxksackievirus: 1) Viral entry through binding coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF), 2) Internalization and transport of viral particles to the Golgi and endoplasmic reticulum (ER) 3) viral uncoating, 4) release of viral RNA, translation of RNA by ribosomes on the rough endoplasmic reticulum (ER) into viral polyprotein, 5) autocleavage of polyprotein into viral structural and functions proteins, 6) positive and negative strand RNA transcription to replicate viral genome, 7) release of viral genome in to the cytosol to encapsidate with

structural proteins, 8) formation and release of viral progeny.

reported that men develop myocarditis twice as often as women and recent clinical work as supported this notion though the incidence is not quite as high as claimed in 1980 (Woodruff 1980; Mason et al. 1995; Kuhl et al. 2003; Cooper 2009).

Diagnosis and treatment of viral myocarditis is challenging due to the lack in specific clinical features and signature serological markers known for the acute phase of disease. The standard Dallas criteria that were typically used for diagnosis of myocarditis are not appropriate for determining viral or autoimmune myocarditis since they avoid the identification of inflammation or viral genome in the heart (Aretz 1987; Aretz et al. 1987). A more appropriate set of guidelines was established in 1995 by the WHO, with a classification of cardiomyopathies requiring endomyocardial biopsy, histological Dallas criteria, immunohistochemistry and viral PCR amongst the criteria for diagnosis (Richardson et al. 1996). Four stages of clinical disease in humans have been described since the implication of the 1995 WHO criteria: fulminant, subacute, chronic active and chronic persistent myocarditis. The first three stages involve mild to moderate dysfunction of the left ventricle, with the fourth, chronic persistent stage, characterized by normal ventricular function. Both chronic stages have ongoing inflammation with the development of scar tissue from myocardial damage. It is only during the chronic persistent stage that viral genome is detected from endomyocardial biopsy tissue (Lieberman et al. 1991; Olsen 1993). The incidence of viral infection, specifically coxsackievirus B infection, in human myocarditis, originates from seroepidemiologic and molecular studies between the 1950s and 1990s and from observations of viral genomes in cardiac tissue more prevalently in dilated cardiomyopathy patients compared to valvular or ischemic cardiomyopathies.

The true etiology and molecular pathogenesis responsible for viral myocarditis in humans remains unclear. Serological studies and endomyocardial biopsies from myocarditis patients have associated over 20 different viruses including coxsackieviruses, adenoviruses, cytomegaloviruses, parvoviruses, influenza viruses, and even human immunodeficiency viruses with the disease (Yajima et al. 2009; Blauwet et al. 2010). Of all the viruses implicated, it is the enteroviruses, specifically coxsackievirus B, which show the most likely contribution.

Coxsackievirus A and B are enteroviruses that are a part of the *Picornaviridae* family (Baboonian et al. 1997). They are human pathogens transmitted fecal-orally that cause enteric diseases (coxsackievirus A) as well as severe disease in the heart, pancreas, and central nervous system (coxsackievirus B) (Baboonian et al. 1997). Coxsackievirus A has 23 different serotypes, whereas coxsackievirus B has six. The six coxsackievirus B serotypes can instigate a variety of diseases including two very important autoimmune diseases: myocarditis and type I diabetes (Richer et al. 2009). It is also important to note that all coxsackievirus B serotypes are capable of triggering systemic disease in infants that can devastating lead to death (Esfandiarei et al. 2008). Relevant to myocarditis is the coxsackievirus B serotype 3 (coxsackievirus B3). Coxsackievirus B3 has been linked to approximately 30% of new dilated cardiomyopathy cases per anum though data establishing a direct link between coxsackievirus B3 pathogenesis and the onset of myocarditis in patients is lacking (Huber et al. 1998). The B3 serotype has a 7.4-kb singlestranded positive-sense RNA genome containing a VPg (3B) protein at the 5' end. The 7 methyl guanosine-like cap influences replication and translation following virus entry (Flanegan et al. 1977). To gain entry in to a cell, coxsackieviruses interact with both coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF) located both in the host cell membrane (Figure 1) (Pelletier et al. 1988).

reported that men develop myocarditis twice as often as women and recent clinical work as supported this notion though the incidence is not quite as high as claimed in 1980

Diagnosis and treatment of viral myocarditis is challenging due to the lack in specific clinical features and signature serological markers known for the acute phase of disease. The standard Dallas criteria that were typically used for diagnosis of myocarditis are not appropriate for determining viral or autoimmune myocarditis since they avoid the identification of inflammation or viral genome in the heart (Aretz 1987; Aretz et al. 1987). A more appropriate set of guidelines was established in 1995 by the WHO, with a classification of cardiomyopathies requiring endomyocardial biopsy, histological Dallas criteria, immunohistochemistry and viral PCR amongst the criteria for diagnosis (Richardson et al. 1996). Four stages of clinical disease in humans have been described since the implication of the 1995 WHO criteria: fulminant, subacute, chronic active and chronic persistent myocarditis. The first three stages involve mild to moderate dysfunction of the left ventricle, with the fourth, chronic persistent stage, characterized by normal ventricular function. Both chronic stages have ongoing inflammation with the development of scar tissue from myocardial damage. It is only during the chronic persistent stage that viral genome is detected from endomyocardial biopsy tissue (Lieberman et al. 1991; Olsen 1993). The incidence of viral infection, specifically coxsackievirus B infection, in human myocarditis, originates from seroepidemiologic and molecular studies between the 1950s and 1990s and from observations of viral genomes in cardiac tissue more prevalently in dilated

(Woodruff 1980; Mason et al. 1995; Kuhl et al. 2003; Cooper 2009).

cardiomyopathy patients compared to valvular or ischemic cardiomyopathies.

both in the host cell membrane (Figure 1) (Pelletier et al. 1988).

contribution.

The true etiology and molecular pathogenesis responsible for viral myocarditis in humans remains unclear. Serological studies and endomyocardial biopsies from myocarditis patients have associated over 20 different viruses including coxsackieviruses, adenoviruses, cytomegaloviruses, parvoviruses, influenza viruses, and even human immunodeficiency viruses with the disease (Yajima et al. 2009; Blauwet et al. 2010). Of all the viruses implicated, it is the enteroviruses, specifically coxsackievirus B, which show the most likely

Coxsackievirus A and B are enteroviruses that are a part of the *Picornaviridae* family (Baboonian et al. 1997). They are human pathogens transmitted fecal-orally that cause enteric diseases (coxsackievirus A) as well as severe disease in the heart, pancreas, and central nervous system (coxsackievirus B) (Baboonian et al. 1997). Coxsackievirus A has 23 different serotypes, whereas coxsackievirus B has six. The six coxsackievirus B serotypes can instigate a variety of diseases including two very important autoimmune diseases: myocarditis and type I diabetes (Richer et al. 2009). It is also important to note that all coxsackievirus B serotypes are capable of triggering systemic disease in infants that can devastating lead to death (Esfandiarei et al. 2008). Relevant to myocarditis is the coxsackievirus B serotype 3 (coxsackievirus B3). Coxsackievirus B3 has been linked to approximately 30% of new dilated cardiomyopathy cases per anum though data establishing a direct link between coxsackievirus B3 pathogenesis and the onset of myocarditis in patients is lacking (Huber et al. 1998). The B3 serotype has a 7.4-kb singlestranded positive-sense RNA genome containing a VPg (3B) protein at the 5' end. The 7 methyl guanosine-like cap influences replication and translation following virus entry (Flanegan et al. 1977). To gain entry in to a cell, coxsackieviruses interact with both coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF) located

Fig. 1. Tentative coxsackievirus life cycle. The general host and virus components suggested to contribute to production of new coxksackievirus: 1) Viral entry through binding coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF), 2) Internalization and transport of viral particles to the Golgi and endoplasmic reticulum (ER) 3) viral uncoating, 4) release of viral RNA, translation of RNA by ribosomes on the rough endoplasmic reticulum (ER) into viral polyprotein, 5) autocleavage of polyprotein into viral structural and functions proteins, 6) positive and negative strand RNA transcription to replicate viral genome, 7) release of viral genome in to the cytosol to encapsidate with structural proteins, 8) formation and release of viral progeny.

Once the virus enters the cytosol and uncoats, its positive-sense genome is released in the cytosol for translation and later, transcription. As a polyprotein comprised of the virus proteins VP4, VP3, VP2, VP, 2A, 2B, 2C, 3A, 3B, 3C, and 3D emerges from translation at the rough endoplasmic reticulum (ER), it is cleaved into its respective structural and functional proteins. The virally encoded 3Dpol is a RNA-dependent RNA polymerase that transcribes viral positive-sense RNA in to negative-sense RNA strands that serve as intermediates for the transcription of multiple positive-sense RNA strands needed for new progeny virions. After synthesis, the newly generated positive-RNA strands are packaged in new virus particles formed by the newly generated structural and functional virus proteins. The new progeny viruses are then released via plasma membrane by a mechanism likely mediated by viral

The Key Players of Coxsackievirus-Induced Myocarditis 247

attributed to low mRNA and protein levels of TNF-α and IL-Iβ as well as reduced CD1d expression on splenic lymphocytes. CD1d is an important non-classical major histocompatibility complex antigen that can be regulated by TNF-α to induce myocarditis

Not only is susceptibility to developing myocarditis dictating by sex, but development of chronic disease depends on the mouse strain. A.BY/SnJ & SWR/J are susceptible mouse strains that can develop ongoing myocarditis, where viral RNA is detected within the myocardium. C57BL/6J & DBA/1J mice are resistant strains capable of eliminating virus just after the early acute phase of disease. Tomioka and colleagues investigated neutralizing antibodies and their role in virus-induced myocarditis and B-cell-mediated immunity using BALB/c mice (Esfandiarei et al. 2008). NK-deficient mice have been used to look at the role of natural killer (NK) cells in killing virus-infected cardiomyocytes (Godeny et al. 1986). Perforin knockout mice inoculated with coxsackievirus B3 have also been used to describe the interplay of virus infection and lymphocyte infiltration in the myocytes and their effect on disease outcome (Godeny et al. 1987). Interestingly, with coxsackievirus B3 infection in suckling, weaning and adolescent mice, coxsackievirus B3 replicates in the heart, pancreas, spleen, and brain and causes human disease-like symptoms. In fact, following IP injection,

In mice, coxsackievirus B3 can induce two forms of inflammatory heart disease, acute only or acute and chronic (biphasic) autoimmune disease (Horwitz et al. 2000; Cunningham 2001; Fairweather et al. 2001). Interestingly, coxsackievirus B3 replication is mainly observed in the pancreas and to a lesser extent in the heart. In genetically susceptible mice, such as A/J2, Balb/c and NOD mice, chronic autoimmune myocarditis after coxsackievirus B3 infection is observed. Autoimmune myocarditis in mice presents as early as day 7 pi with inflammatory cell infiltration in the heart and the formation of multifocal inflammatory lesions. At this stage, autoantibodies (autoAbs) against heart antigens, like cardiac myosin (cardiac myosin), are seen. In NOD mice, isotype switching ensues after 2 to 3 weeks, with the autoAbs switching from IgM to IgG subclasses (Kaya et al. 2001; Kaya et al. 2002). It is particularly remarkable to note that the chronic autoimmune heart disease induced by coxsackievirus B3 in mice resembles inflammatory heart disease seen with myocarditis and dilated cardiomyopathy in humans. There are still many complications with the full characterization and study of chronic virus and/or autoimmune induced myocarditis in mice and humans. One such complication is the

As described in the previous section, mouse models greatly aid the analysis of autoimmune diseases. To set apart the autoimmune phase of disease from acute infection an experimental induced autoimmune myocarditis model was developed (Blyszczuk et al. 2008). Experimental induced autoimmune myocarditis mimics the typical chronic phase of disease observed in genetically susceptible mice infected with coxsackievirus B and different stages of disease severity observed with experimental induced autoimmune myocarditis models are graded according to the extent of inflammatory infiltrates at the peak of inflammation. Autoimmune myocarditis can be induced by the injection of cardiac myosin with complete Freund's adjuvant and pertussis toxin. Mice injected with this combination of self- antigen and adjuvants are able to generate cardiac myosin-specific autoantibodies and present with

three distinct immunovirological phases of disease have been observed.

susceptibility in female mice (Huber 2010).

acute infection that precedes chronic disease.

**3. Experimental autoimmune myocarditis** 

protein 2B (van Kuppeveld et al. 1997; Esfandiarei et al. 2008). Though many groups have identified key virus and host interactions throughout the coxsackievirus B life cycle, there still remains many stages unsolved. Nevertheless, the role of coxsackievirus B in the onset of myocarditis has been extensively studied thus far in animal models and cell culture systems. Here, we will discuss clinical and mouse studies that have investigated coxsackievirusinduced myocarditis and the role of key immune players in disease pathogenesis.
