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1.  High prevalence of enteroviral genomic sequences in myocardium from cases of endemic cardiomyopathy (Keshan disease) in China 
Heart  2000;83(6):696-701.
OBJECTIVE—To verify the aetiological involvement of enterovirus and identify the viral genomic sequences in Keshan disease.
DESIGN—Formalin fixed, paraffin embedded myocardial necropsy tissue samples were collected in Keshan disease endemic regions. Fourteen cases with a histologically confirmed diagnosis of subacute or chronic Keshan disease were studied. Control tissue included 10 samples of myocardium from cases of cerebral trauma and one from accidental acid intoxication. One sample from a case of enteroviral myocarditis was used as a positive control. The presence of viral genomic RNA was investigated using an established reverse transcription nested polymerase chain reaction (PCR) coupled with direct nucleotide sequencing. Further investigations of PCR positive samples included in situ antigen detection or hybridisation to confirm positive results.
RESULTS—Nine of 14 myocardial samples from Keshan disease cases and the positive control were positive for the enteroviral RNA. All the controls were negative. Six of the PCR positive samples were investigated further by in situ enteroviral antigen or RNA detection and all were positive. DNA sequencing of six representative PCR products confirmed that they were homologous to the 5' non-translated region of enteroviral genomic RNA. Five had highest homology to coxsackievirus B genotypes and one was identical to poliovirus type 3.
CONCLUSIONS—These results support an aetiological role for enteroviral infection in Keshan disease. Nucleotide sequence data suggest that coxsackievirus B or coxsackie B like viruses are often involved in Keshan disease.


Keywords: enterovirus; coxsackievirus; cardiomyopathy; Keshan disease
doi:10.1136/heart.83.6.696
PMCID: PMC1760852  PMID: 10814633
2.  Characterization of Enterovirus Isolates from Patients with Heart Muscle Disease in a Selenium-Deficient Area of China 
Journal of Clinical Microbiology  2000;38(10):3538-3543.
An association of enterovirus infection with endemic cardiomyopathy (Keshan disease [KD]) and outbreaks of myocarditis in selenium-deficient rural areas of southwestern China has been established. Enteroviruses have been isolated from patients with KD or during outbreaks of myocarditis in last two decades. Six of these isolates grew readily in cell lines (Vero or HEp-2) and were investigated by a novel molecular typing method apart from serotyping and pathogenicity. A neutralization assay identified two isolates from KD as coxsackievirus serotype B2 (CVB2) and two isolates from myocarditis as coxsackievirus serotype B6 (CVB6) but failed to type the remaining two isolates, also from myocarditis. Direct nucleotide sequencing of reverse transcription-PCR products amplified from the 5′ nontranslated region (5′NTR) of these viruses confirmed that they belong to a phylogenetic cluster consisting of coxsackie B-like viruses, including some echovirus serotypes. Sequence analysis of the coding region for viral capsid protein VP1 showed that two isolates serotyped as CVB2 have the highest amino acid sequence homology with CVB2 and that the remaining four isolates, two CVB6 and the two unknown serotypes, are most closely related to the sequence of CVB6. Sequences among these isolates varied from 82.3 to 99% in the 5′NTR and from 69 to 99% in VP1, indicating no cross contamination. The pathogenicity of these viruses in adult and suckling mice was assessed. None caused pathologic changes in the hearts of adult MF-1 or SWR mice, although pancreatitis was evident. However, the four CVB6-like viruses caused death in suckling mice, similar to a virulent coxsackievirus group B3 laboratory strain. In conclusion, the sequence data confirm that coxsackievirus group B serotypes are predominant in the region in which KD is endemic and may be the etiological agents in outbreaks of myocarditis. VP1 genotyping of enteroviruses is accurate and reliable. Animal experiments indicate that isolates may differ in pathogenicity.
PMCID: PMC87433  PMID: 11015360
3.  Coxsackievirus B3 sequences in the myocardium of fatal cases in a cluster of acute myocarditis in Greece 
Journal of Clinical Pathology  2005;58(4):357-360.
Aim: The investigation of three fatal cases during a nationwide cluster of cases of an upper respiratory tract infection (URTI) associated with myocarditis and/or pericarditis in Greece in 2002.
Methods: In the three women who died, necropsies were performed and tissue sections were taken for histological examination, antigen detection by immunohistochemistry and indirect immunofluorescence assay (IFA), amplification of viral genomes by nested reverse transcription polymerase chain reaction (RT-PCR), and sequence analysis.
Results: All samples showed histological signs of active myocarditis. Immunohistochemistry revealed the presence of the enterovirus VP1 family of proteins and IFA revealed the presence of coxsackievirus B3 antigen. Nested RT-PCR amplified enteroviral alleles of the 5′-untranslated region which were identical to each other and to the coxsackievirus B3 sequences.
Conclusions: This study provides pathological evidence of enteroviral infection among fatal myocarditis cases in a nationwide URTI cluster of cases associated with myocarditis and/or pericarditis.
doi:10.1136/jcp.2004.020099
PMCID: PMC1770614  PMID: 15790697
acute myocarditis; enterovirus; immunofluorescence; immunohistochemistry; sequencing
4.  No detection of enteroviral genome in the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy 
Journal of Clinical Pathology  2000;53(5):382-387.
Aims—Despite the evidence of familial occurrence, chromosomal gene mapping, and apoptosis as a mechanism of myocyte death, the aetiopathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) remains speculative. Because of the frequent histological finding of focal inflammatory infiltrates, the hypothesis of an infective myocarditis aetiology has been put forward. The aim of this investigation was to test this hypothesis. The presence of enteroviruses was investigated by a highly sensitive and specific molecular technique.
Methods—Endomyocardial tissue samples from 20 patients with ARVC (11 male, nine female; mean age, 40 years; SD, 16) and 20 control subjects with other cardiac diseases were analysed using reverse transcription and nested polymerase chain reaction (PCR). Myocardial samples obtained from four patients with enteroviral myocarditis and coxsackie B3 virus infected cells were used as positive controls.
Results—Endomyocardial biopsy was diagnostic for ARVC in all patients: myocardial atrophy was seen, with less than 45% residual myocytes. Foci of inflammatory infiltrates were seen in four biopsies, and the cells were identified by immunohistochemistry as mainly T cells. All samples, from both patients with ARVC and subjects with other cardiac diseases, were negative for enteroviral genome by means of nested PCR.
Conclusion—These findings indicate that enteroviruses are not involved in the aetiopathogenesis of ARVC. Future molecular studies should investigate the presence of other infective agents, as well as their possible role in triggering apoptosis.
Key Words: arrhythmogenic right ventricular cardiomyopathy • myocarditis • enterovirus • molecular biology
doi:10.1136/jcp.53.5.382
PMCID: PMC1731194  PMID: 10889821
5.  Analysis of sudden unexpected death in southern Ontario, with emphasis on myocarditis. 
The records of all 2427 autopsies performed at the Brantford (Ont.) General and Paris (Ont.) Willett hospitals from Jan. 1, 1969 to Aug. 15, 1978 were reviewed. Of the 1299 cases of sudden unexpected death investigated by a coroner almost 28% were due to unnatural causes--violence or poisoning. The main cause of natural sudden death was coronary artery disease, which accounted for 43.3% of all the sudden unexpected deaths. In 20 cases the cause of death was thought to be viral myocarditis, and in 9 of the 20 there was serologic evidence of at least previous coxsackievirus disease. Two of the nine cases were of special interest because of the finding of giant-cell myocarditis in one and aortic valve disease in the other. Eleven of the 20 persons were aged 13 to 46 years. These findings support the view that the most serious manifestation of enterovirus infection today is cardiac damage by coxsackieviruses.
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PMCID: PMC1819176  PMID: 436050
6.  Coxsackievirus B detection in cases of myocarditis, myopericarditis, pericarditis and dilated cardiomyopathy in hospitalized patients 
Molecular Medicine Reports  2014;10(6):2811-2818.
Coxsackieviruses B (CV-B) are known as the most common viral cause of human heart infections. The aim of the present study was to assess the potential role of CV-B in the etiology of infectious heart disease in hospitalized patients. The present study is based on blood, pericardial fluid and heart biopsies from 102 patients and 100 control subjects. All of the samples were examined for the detection of specific enteroviral genome using the reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis. Immunohistochemical investigations for the detection of the enteroviral capsid protein, VP1, from the biopsies were performed. The samples were cultured on confluent KB monolayer cell line for possible virus isolation. The epidemiological data were also collected. CV-B was detected in 28 of the 102 patients. The sequence analysis demonstrated that 27 strains were identical to CV-B3 and only one strain was identical to CV-B1. Furthermore, VP1 in the heart biopsies was detected in enterovirus-positive cases, as revealed by RT-PCR. Pericarditis infection was more frequent than myocarditis (P<0.05) or myopericarditis (P=0.05). The epidemiological data demonstrate that CV-B heart infections occur mainly during autumn and winter, and young male adults are more susceptible than adolescents or adults (P<0.5). The present findings demonstrate a higher prevalence of viral heart infections, suggesting that CV-B may significantly contribute to heart infections.
doi:10.3892/mmr.2014.2578
PMCID: PMC4227425  PMID: 25241846
coxsackievirus B; human heart infections; molecular diagnosis; immunohistochemical investigations; epidemiology
7.  From gene expression profiles to biological validation in enteroviral heart disease 
In humans, coxsackievirus B3 is the primary etiological agent of viral myocarditis, an inflammatory disease process involving the heart muscle. Specific therapy is currently unavailable. Viral myocarditis is a complex, multiphasic infectious-inflammatory-reparative process. To address the temporal dimensionality of myocarditis, array- and nonarray-based molecular techniques, and histological and functional assays were used to help define enteroviral pathogenesis and its relation to heart failure. The application of high throughput genomic strategies and bioinformatics tools – coupled with established molecular techniques – have allowed us to perform a large-scale analysis of gene expression to better understand the host response to viral infection. Differential messenger RNA display, spotted complementary DNA arrays and Affymetrix Gene Chips (Affymetrix, United States) were used to study murine hearts during acute viremic, inflammatory and reparative stages. The observed global decreases in expression of metabolic and mitochondrial genes were focused on. The authors have previously characterized the role of mitochondria-triggered apoptosis, and pro- and anti-apoptotic Bcl-2 family proteins in enteroviral infections. The impact of altered mitochondrial transcripts on such host cell death and on metabolic injury to the heart is currently under study. In the authors’ experience, the experimental progression from high throughput, unbiased analysis to biological validation has been only partially systematic. Insights are offered into the logic behind the selection of genes of potential interest for further investigation in the myocarditis model. A series of criteria for validatory decision-making, which the authors have developed based on their experiences, is described. Such criteria reflect known or putative gene function and expression patterns, as well as pragmatic considerations in the determination of steps toward investigation. This approach may help other investigators who need to dissect large genomic data sets to find targets for biological confirmation. Together, the authors’ genomic studies have generated new, testable hypotheses regarding the interaction between host and enterovirus.
PMCID: PMC2716272  PMID: 19641703
Bioinformatics; Genomics; Microarrays; Myocarditis
8.  Myocarditis, Disseminated Infection, and Early Viral Persistence Following Experimental Coxsackievirus B Infection of Cynomolgus Monkeys 
PLoS ONE  2013;8(9):e74569.
Coxsackievirus B (CVB) infection is a common cause of acute viral myocarditis. The clinical presentation of myocarditis caused by this enterovirus is highly variable, ranging from mildly symptoms to complete hemodynamic collapse. These variations in initial symptoms and in the immediate and long term outcomes of this disease have impeded development of effective treatment strategies. Nine cynomolgus monkeys were inoculated with myocarditic strains of CVB. Virological studies performed up to 28 days post-inoculation demonstrated the development of neutralizing antibody in all animals, and the presence of CVB in plasma. High dose intravenous inoculation (n = 2) resulted in severe disseminated disease, while low dose intravenous (n = 6) or oral infection (1 animal) resulted in clinically unapparent infection. Transient, minor, echocardiographic abnormalities were noted in several animals, but no animals displayed signs of significant acute cardiac failure. Although viremia rapidly resolved, signs of myocardial inflammation and injury were observed in all animals at the time of necropsy, and CVB was detected in postmortem myocardial specimens up to 28 days PI. This non-human primate system replicates many features of illness in acute coxsackievirus myocarditis and demonstrates that myocardial involvement may be common in enteroviral infection; it may provide a model system for testing of treatment strategies for enteroviral infections and acute coxsackievirus myocarditis.
doi:10.1371/journal.pone.0074569
PMCID: PMC3767629  PMID: 24040287
9.  Similar prevalence of enteroviral genome within the myocardium from patients with idiopathic dilated cardiomyopathy and controls by the polymerase chain reaction 
British Heart Journal  1992;68(6):554-559.
Objective—To assess the prevalence and significance of enteroviral genome within myocardial biopsy specimens taken from patients with idiopathic dilated cardiomyopathy and from controls.
Design—Prospective evaluation of myocardial tissue for the presence of an enteroviral genome by the polymerase chain reaction.
Setting—A tertiary referral centre for patients with idiopathic dilated cardiomyopathy.
Patients—Tissue for the study came from 50 consecutive patients with dilated cardiomyopathy, 41 with other forms of heart disease and 34 from coroners' necropsy cases.
Results—Enteroviral genome was detected in 6/50 (12%) patients with dilated cardiomyopathy and 13/75 (17%) of the controls (not significant). No differences were seen between dilated cardiomyopathy patients with or without myocardial enteroviral genome in respect of age; duration of symptoms; proportion of patients with a premorbid acute viral illness, excess alcohol consumption, or hypertension; New York Heart Association functional class; measures of left ventricular function; or endomyocardial histology. Within the control group enteroviral genome was detected in 3/15 (20%) patients with ischaemic heart disease, 2/19 (10·5%) with valvar heart disease, 1/5 (20%) with specific heart muscle disease, 0/2 (0%) with congenital heart disease, and 7/34 (20·6%) cases of sudden death. During 2–52 month follow up (mean 22) 15/44 (34%) patients without myocardial enteroviral genome and 2/6 (33%) with myocardial enteroviral genome died suddenly or required orthotopic heart transplantation for progressive heart failure.
Conclusions—These findings do not support the hypothesis that persistent enteroviral infection is of pathogenic or prognostic importance in dilated cardiomyopathy but they are consistent with enterovirus being a common environmental pathogen.
PMCID: PMC1025684  PMID: 1334684
10.  Serological and molecular evidence of enterovirus infection in patients with end-stage dilated cardiomyopathy. 
Heart  1996;76(3):243-249.
OBJECTIVE: To study the relative diagnostic value of enterovirus-specific molecular biological and serological assays in patients with end-stage dilated cardiomyopathy, and to investigate the possible role of other cardiotropic viruses in dilated cardiomyopathy. DESIGN: Analysis of recipient myocardial tissue and serum from patients with dilated cardiomyopathy and controls undergoing cardiac transplantation for end-stage cardiac disease. SETTING: University virology department and transplantation unit. METHODS: Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis of myocardial RNA and DNA; enterovirus-specific in situ hybridization; enterovirus-specific immunoglobulin M detection. RESULTS: Enterovirus RNA was detected in myocardial tissue from only a small proportion of (five of 75) hearts. However, although enterovirus-specific immunoglobulin M responses were detected in 22 (28%) of 39 controls patients, a significantly higher prevalence was observed among patients with dilated cardiomyopathy (22 (56%) of 39 patients; P < 0.005). All enteroviruses detected in myocardium showed greatest nucleotide sequence homology with coxsackievirus type B3. Detection of enterovirus RNA in myocardium by the polymerase chain reaction and by in situ hybridisation gave comparable results. Other potentially cardiotropic virus genomes, including human cytomegalovirus, influenzaviruses, and coronaviruses were not detected in myocardium. CONCLUSION: This study found that enterovirus-specific immunoglobulin M responses provided the strongest evidence of enterovirus involvement in patients with end-stage dilated cardiomyopathy. However, the high background prevalence of these responses limits their diagnostic value. The finding that enteroviruses detected in myocardium were coxsackievirus type B3 accords with recent findings in patients with acute myocarditis, and indicates that this serotype is the major cardiotropic human enterovirus.
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PMCID: PMC484515  PMID: 8868984
11.  Molecular detection and serotypic analysis of enterovirus RNA in archival specimens from patients with acute myocarditis. 
British Heart Journal  1995;74(5):522-527.
OBJECTIVE--To determine whether enterovirus RNA can be demonstrated in archival necropsy material in acute myocarditis. DESIGN--Analysis of paraffin embedded myocardial tissue from cases of acute myocarditis. SETTING--University virology department. METHODS--Extraction of RNA from tissue followed by polymerase chain reaction (PCR) and DNA sequence analysis. PATIENTS--Six patients with histologically proven myocarditis and eight controls. RESULTS--Enterovirus RNA was identified in 5 of 6 patients with myocarditis and in none of the controls. The nucleotide sequences of the PCR products showed greatest similarity to group B coxsackieviruses, particularly coxsackievirus B3. CONCLUSION--This study indicates that archival tissue samples, even histologically stained tissue sections, can be used to study the role of enteroviruses in myocardial disease using molecular detection techniques. If a predominant role for coxsackievirus B3 in myocarditis is confirmed by further study, this may have implications for the development of a specific vaccine.
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PMCID: PMC484072  PMID: 8562237
12.  5' terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart 
Virology  2008;375(2):480-491.
Enteroviruses can induce human myocarditis, which can be modeled in mice inoculated with group B coxsackieviruses (CVB) and in which CVB evolve to produce defective, terminally deleted genomes. The 5' non-translated region (NTR) was enzymatically amplified from heart tissue of a fatal case of enterovirus-associated myocarditis in Japan in 2002. While no intact 5' viral genomic termini were detected, 5' terminal deletions ranged in size from 22−36 nucleotides. Sequence of the 5' third of this viral genome is of a modern strain, closely related to CVB2 strains isolated in Japan in 2002. A CVB3 chimera containing the 5’ NTR with a 22 nt deletion produced progeny virus upon transfection of HeLa cells. When the 5' 22 nucleotide deletion was repaired, the virus induced myocarditis in mice and replicated like wild-type virus in murine heart cells. This is the first report of these naturally occurring defective enteroviral genomes in human myocarditis.
doi:10.1016/j.virol.2008.02.030
PMCID: PMC2440640  PMID: 18378272
coxsackievirus; human myocarditis; defective enterovirus; 5’ terminal deletion
13.  In Vivo Ablation of Type I Interferon Receptor from Cardiomyocytes Delays Coxsackieviral Clearance and Accelerates Myocardial Disease 
Journal of Virology  2014;88(9):5087-5099.
ABSTRACT
Acute coxsackievirus B3 (CVB3) infection is one of the most prevalent causes of acute myocarditis, a disease that frequently is identified only after the sudden death of apparently healthy individuals. CVB3 infects cardiomyocytes, but the infection is highly focal, even in the absence of a strong adaptive immune response, suggesting that virus spread within the heart may be tightly constrained by the innate immune system. Type I interferons (T1IFNs) are an obvious candidate, and T1IFN receptor (T1IFNR) knockout mice are highly susceptible to CVB3 infection, succumbing within a few days of challenge. Here, we investigated the role of T1IFNs in the heart using a mouse model in which the T1IFNR gene can be ablated in vivo, specifically in cardiomyocytes. We found that T1IFN signaling into cardiomyocytes contributed substantially to the suppression of viral replication and infectious virus yield in the heart; in the absence of such signaling, virus titers were markedly elevated by day 3 postinfection (p.i.) and remained high at day 12 p.i., a time point at which virus was absent from genetically intact littermates, suggesting that the T1IFN-unresponsive cardiomyocytes may act as a safe haven for the virus. Nevertheless, in these mice the myocardial infection remained highly focal, despite the cardiomyocytes' inability to respond to T1IFN, indicating that other factors, as yet unidentified, are sufficient to prevent the more widespread dissemination of the infection throughout the heart. The absence of T1IFN signaling into cardiomyocytes also was accompanied by a profound acceleration and exacerbation of myocarditis and by a significant increase in mortality.
IMPORTANCE Acute coxsackievirus B3 (CVB3) infection is one of the most common causes of acute myocarditis, a serious and sometimes fatal disease. To optimize treatment, it is vital that we identify the immune factors that limit virus spread in the heart and other organs. Type I interferons play a key role in controlling many virus infections, but it has been suggested that they may not directly impact CVB3 infection within the heart. Here, using a novel line of transgenic mice, we show that these cytokines signal directly into cardiomyocytes, limiting viral replication, myocarditis, and death.
doi:10.1128/JVI.00184-14
PMCID: PMC3993796  PMID: 24574394
14.  Prevalence of toxoplasma myocarditis in patients with the acquired immunodeficiency syndrome. 
British Heart Journal  1993;70(4):376-381.
OBJECTIVE--To evaluate the prevalence of cardiac toxoplasmosis in a series of 182 necropsies performed between 1987 and 1991 on patients infected with the human immunodeficiency virus (HIV), to correlate this prevalence with the ante mortem diagnosis of cardiac involvement, and to assess the role of such cardiac lesions in the immediate cause of death. PATIENTS AND METHODS--Complete necropsies of 182 HIV-infected patients (48 women, 134 men) were performed consecutively between 1987 and 1991. Risk factors, identified in 174 cases, included drug abuse (111/182), homosexuality (51/182), and blood transfusions (12/182). 16 samples were systematically obtained from each heart for histological study. If trophozoites or lymphocytic myocarditis were seen, immunohistochemical investigations were carried out with polyclonal antibodies for Toxoplasma gondii. An ultrastructural study was performed in four patients with toxoplasma myocarditis. Myocardial lesions were defined by the Dallas classification. Clinical data (and information on electrocardiograms and echocardiograms) were obtained from medical records. RESULTS--Cardiac toxoplasmosis was diagnosed at necropsy in 21 (12%) patients. Cardiac lesions were associated with toxoplasmic encephalitis in 18 patients and were solitary in three patients. Acute diffuse myocarditis was present in 6/21, rare foci of myocarditis were seen in 8/21, and intramyocytic toxoplasmic cysts without any inflammatory reaction or necrosis were seen in 4/21. Anti-toxoplasma immunolabelling showed cardiac toxoplasmosis in three patients with lymphocytic myocarditis. Particles with the ultrastructural characteristics of Toxoplasma gondii trophozoites were seen in four cases. Six patients had presented with cardiac symptoms, confirmed by electrocardiographic and echocardiographic abnormalities during their disease course, and their cardiac lesions were directly responsible for the death. CONCLUSION--Cardiac toxoplasmosis was common in this necropsy series of HIV-infected patients. Cardiac toxoplasmosis had been suspected clinically in four patients. Myocardial lesions were generally asymptomatic and were not discovered until necropsy. Solitary cardiac involvement was not uncommon reflecting parasite reactivation at a myocardial site. The incidence of cardiac toxoplasmosis in this group of immunodepressed subjects from an area with a high prevalence of this parasitic disease justifies regular follow up of such patients by electrocardiography and echocardiography as well as immediate administration of anti-toxoplasma treatment should sudden heart failure occur.
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PMCID: PMC1025336  PMID: 8217449
15.  Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers 
PLoS Pathogens  2014;10(4):e1004045.
Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.
Author Summary
Enteroviruses are significant human pathogens, causing myocarditis, aseptic meningitis and encephalitis. The mechanisms of enterovirus dissemination in the host and cell-to-cell spread may be critical factors influencing viral pathogenesis. Here, we have generated a recombinant coxsackievirus expressing “fluorescence timer” protein (Timer-CVB3) which assists in following the progression of infection within the host. Unexpectedly, we observed the shedding of microvesicles containing virus in partially-differentiated progenitor cells infected with Timer-CVB3. These extracellular microvesicles (EMVs) were released in high levels following cellular differentiation, and may play a role in virus dissemination. Timer-CVB3 will be a valuable tool in monitoring virus spread in the infected host.
doi:10.1371/journal.ppat.1004045
PMCID: PMC3983045  PMID: 24722773
16.  Autoimmune Myocarditis, Valvulitis, and Cardiomyopathy 
Cardiac myosin-induced autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by CD4+ T cells (Smith and Allen 1991; Li, Heuser et al. 2004). It is a paradigm of the immune-mediated cardiac damage believed to play a role in the pathogenesis of a subset of postinfectious human cardiomyopathies (Rose, Herskowitz et al. 1993). Myocarditis is induced in susceptible mice by immunization with purified cardiac myosin (Neu, Rose et al. 1987) or specific peptides derived from cardiac myosin (Donermeyer, Beisel et al. 1995; Pummerer, Luze et al. 1996) (see Basic Protocol 1), or by adoptive transfer of myosin-reactive T cells (Smith and Allen 1991) (see Alternate Protocol). Myocarditis has been induced in Lewis rats by immunization with purified rat or porcine cardiac myosin (Kodama, Matsumoto et al. 1990; Li, Heuser et al. 2004) (see Basic Protocol 2) or S2-16 peptide (Li, Heuser et al. 2004), or by adoptive transfer of T cells stimulated by specific peptides derived from cardiac myosin (Wegmann, Zhao et al. 1994). Myocarditis begins 12 to 14 days after the first immunization, and is maximal after 21 days.
Other animal models commonly used to study myocarditis development include the pathogen-induced models in which disease is initiated by viral infection. The first murine model of acute viral myocarditis causes sudden death via viral damage to cardiomyocytes (Huber, Gauntt et al. 1998; Horwitz, La Cava et al. 2000; Fong 2003; Fuse, Chan et al. 2005; Fairweather and Rose 2007; Cihakova and Rose 2008) whereas the second model is based on inoculation with heart-passaged coxsackievirus B3 (CVB3) that includes damaged heart proteins (Fairweather, Frisancho-Kiss et al. 2004; Fairweather D 2004; Fairweather and Rose 2007; Cihakova and Rose 2008)
In addition to the protocols used to induce EAM in mice and rats, support protocols are included for preparing purified cardiac myosin using mouse or rat heart tissue (see Support Protocol 1), preparing purified cardiac myosin for injection (see Support Protocol 2), and collecting and assessing hearts by histopathological means (see Support Protocol 3).
doi:10.1002/0471142735.im1514s101
PMCID: PMC3672855  PMID: 23564686
17.  Direct Gene Transfer with IP-10 Mutant Ameliorates Mouse CVB3-Induced Myocarditis by Blunting Th1 Immune Responses 
PLoS ONE  2011;6(3):e18186.
Background
Myocarditis is an inflammation of the myocardium that often follows the enterovirus infections, with coxsackievirus B3 (CVB3) being the most dominant etiologic agent. We and other groups previously reported that chemokine IP-10 was significantly induced in the heart tissue of CVB3-infected mice and contributed to the migration of massive inflammatory cells into the myocardium, which represents one of the most important mechanisms of viral myocarditis. To evaluate the direct effect of IP-10 on the inflammatory responses in CVB3 myocarditis, herein an IP-10 mutant deprived of chemo-attractant function was introduced into mice to antagonize the endogenous IP-10 activity, and its therapeutic effect on CVB3-induced myocarditis was evaluated.
Methodology/Principal Findings
The depletion mutant pIP-10-AT, with an additional methionine after removal of the 5 N-terminal amino acids, was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection. Compared with vector or no treatment, pIP-10-AT treatment had significantly reduced heart/body weight ratio and serum CK-MB level, increased survival rate and improved heart histopathology, suggesting an ameliorated myocarditis. This therapeutic effect was not attributable to an enhanced viral clearance, but to a blunted Th1 immune response, as evidenced by significantly decreased splenic CD4+/CD8+IFN-γ+ T cell percentages and reduced myocardial Th1 cytokine levels.
Conclusion/Significance
Our findings constitute the first preclinical data indicating that interfering in vivo IP-10 activity could ameliorate CVB3 induced myocarditis. This strategy may represent as a new therapeutic approach in treating viral myocarditis.
doi:10.1371/journal.pone.0018186
PMCID: PMC3062568  PMID: 21445362
18.  Impairment of Immunoproteasome Function by β5i/LMP7 Subunit Deficiency Results in Severe Enterovirus Myocarditis 
PLoS Pathogens  2011;7(9):e1002233.
Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits β1i/LMP2, β2i/MECL-1 and β5i/LMP7 forming an alternative proteasome isoform, the immunoproteasome (IP). The in vivo function of IPs in pathogen-induced inflammation is still a matter of controversy. IPs were mainly associated with MHC class I antigen processing. However, recent findings pointed to a more general function of IPs in response to cytokine stress. Here, we report on the role of IPs in acute coxsackievirus B3 (CVB3) myocarditis reflecting one of the most common viral disease entities among young people. Despite identical viral load in both control and IP-deficient mice, IP-deficiency was associated with severe acute heart muscle injury reflected by large foci of inflammatory lesions and severe myocardial tissue damage. Exacerbation of acute heart muscle injury in this host was ascribed to disequilibrium in protein homeostasis in viral heart disease as indicated by the detection of increased proteotoxic stress in cytokine-challenged cardiomyocytes and inflammatory cells from IP-deficient mice. In fact, due to IP-dependent removal of poly-ubiquitinylated protein aggregates in the injured myocardium IPs protected CVB3-challenged mice from oxidant-protein damage. Impaired NFκB activation in IP-deficient cardiomyocytes and inflammatory cells and proteotoxic stress in combination with severe inflammation in CVB3-challenged hearts from IP-deficient mice potentiated apoptotic cell death in this host, thus exacerbating acute tissue damage. Adoptive T cell transfer studies in IP-deficient mice are in agreement with data pointing towards an effective CD8 T cell immune. This study therefore demonstrates that IP formation primarily protects the target organ of CVB3 infection from excessive inflammatory tissue damage in a virus-induced proinflammatory cytokine milieu.
Author Summary
The proteasome recognizes and degrades protein substrates tagged with poly-ubiquitin chains. Immune cells and cells activated by inflammatory cytokines/interferons express immunoproteasomes (IPs) that are characterized by unique catalytic subunits with increased substrate turnover. In infectious disease, the function of IPs is still a matter of controversial debate. Here, we report on a novel innate function of IPs in viral infection. We studied the murine model of acute enterovirus myocarditis, which represents one of the most common viral disease entities among young people. We found that IPs protect the pathogen-challenged tissue from severe injury, which was reflected in severe myocardial destruction and large inflammatory foci in mice lacking IPs. We show data that this prevention of excessive inflammatory tissue damage in viral heart disease is primarily attributed to preservation of protein homeostasis due to accelerated substrate turnover by IPs. Thus, a major innate function of IPs in viral infection is to stabilize cell viability in inflammatory tissue injury.
doi:10.1371/journal.ppat.1002233
PMCID: PMC3164653  PMID: 21909276
19.  Myocarditis mimicking acute myocardial infarction: role of endomyocardial biopsy in the differential diagnosis 
Heart  2000;84(3):245-250.
OBJECTIVE—To test the hypothesis, using endomyocardial biopsies, that unexplained cases of apparent acute myocardial infarction were caused by myocarditis.
MATERIAL—Between 1992 and 1998, 12 patients were admitted to the coronary care unit with severe chest pain, ST segment elevation, increased serum creatine kinase and MB isoenzyme, and with wall motion abnormalities on echocardiogram highly suggestive of acute myocardial infarction. These patients were further investigated by endomyocardial biopsy, as their coronary angiograms were normal. A diagnosis of myocarditis was made according to the Dallas criteria. A panel of antibodies was used for immunohistochemical characterisation of inflammatory cell infiltrate. Polymerase chain reaction (PCR) was used to detect viral genomes in seven cases.
RESULTS—Haematoxylin and eosin staining of the endomyocardial biopsy showed active myocarditis in six patients and borderline myocarditis in one. Immunohistochemistry was positive for inflammatory cell infiltrates in 11 patients, including all the seven who were positive on haematoxylin and eosin staining according to the Dallas criteria. Only one patient had no evidence of inflammation. PCR was positive in two patients, both for Epstein-Barr virus. Follow up showed complete resolution of echocardiographic abnormalities in all patients except one.
CONCLUSIONS—Myocarditis can mimic acute myocardial infarction in patients with angiographically normal coronary arteries, leading to errors of treatment. In patients with apparent myocardial infarction and a normal coronary angiogram, endomyocardial biopsy may help in the diagnosis of myocarditis. The sensitivity of endomyocardial biopsy was enhanced by using immunohistochemical and molecular biological techniques.


Keywords: acute myocardial infarction; endomyocardial biopsy; myocarditis
doi:10.1136/heart.84.3.245
PMCID: PMC1760950  PMID: 10956283
20.  Echovirus 22 is an atypical enterovirus. 
Journal of Virology  1990;64(6):2692-2701.
Although echovirus 22 (EV22) is classified as an enterovirus in the family Picornaviridae, it is atypical of the enterovirus paradigm, typified by the polioviruses and the coxsackie B viruses. cDNA reverse transcribed from coxsackievirus B3 (CVB3) RNA does not hybridize to genomic RNA of EV22, and conversely, cDNA made to EV22 does not hybridize to CVB3 genomic RNA or to molecular clones of CVB3 or poliovirus type 1. EV22 cDNA does not hybridize to viral RNA of encephalomyocarditis virus or to a molecular clone of Theiler's murine encephalomyelitis virus, members of the cardiovirus genus. The genomic RNA of EV22 cannot be detected by the polymerase chain reaction using generic enteroviral primers. EV22 does not shut off host cell protein synthesis, and the RNA of EV22 is efficiently translated in vitro in rabbit reticulocyte lysates. Murine enterovirus-immune T cells recognize and proliferate against EV22 as an antigen in vitro, demonstrating that EV22 shares an epitope(s) common to enteroviruses but not found among other picornaviruses.
Images
PMCID: PMC249448  PMID: 2159539
21.  General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections. 
Journal of Clinical Microbiology  1992;30(1):160-165.
The aim of this study was to determine the applicability of the polymerase chain reaction (PCR) for routine diagnostic use and for the detection of persistent enteroviral infections. To this end, general primers were selected in the highly conserved part of the 5'-noncoding region of the enteroviral genome. They were tested on 66 different enterovirus serotypes. A specific fragment was amplified from 60 of 66 serotypes. An amplification product was not observed from coxsackievirus types A11, A17, and A24 and echovirus types 16, 22, and 23. Enteroviral RNA was detected by the PCR in routinely collected throat swabs and stool specimens that were found to be positive for enterovirus by isolation in tissue culture. Enteroviral RNA was detected in one of five myocardial biopsy specimens from patients with dilated cardiomyopathy, implicating virus persistence. No amplification product was obtained from eight control samples. Our results demonstrate the significance of the PCR for the detection of enteroviral RNA and, in particular, for the demonstration of persistent enteroviral infections.
Images
PMCID: PMC265013  PMID: 1370845
22.  The whole genome sequence of Coxsackievirus B3 MKP strain leading to myocarditis and its molecular phylogenetic analysis 
Virology Journal  2014;11:33.
Background
In recent years, the reported infection cases by coxsackievirus (CV) have been on the rise. In order to reveal the relationship between the nucleotide and amino acid sequences and the viral virulence of the CVB3/MKP strain causing myocarditis, we initially confirmed the virulence of the strain in myocardial tissue and then carried out the whole genome sequencing of CVB3/MKP strain and performed a phylogenetic analysis among different CVB3 strains.
Methods
CVB3/MKP infected mouse model was established to check lesions of myocardial tissue in mice using immunohistochemical detection at different periods. RT-PCR analysis was used to amplify seven fragments covering the whole viral sequence and comparable analysis was performed.
Results
The immunohistochemical results showed that particles of CVB3/MKP virus persisted in the cardiac tissue and caused severe pathology. The length of whole genome sequence of CVB3/MKP strain was 7400 bp. CVB3/MKP had 99.7% and 99.6% homology in nucleotide sequence with CVB3/28 and non-virulent CVB3/0, respectively. The former can induce pancreatitis and myocarditis. The nucleotide sequence in the 5′untranslated region of CVB3/MKP strain shared 99.6% and 99.5% homology with CVB3/20 and CVB3/Nancy, respectively.
Conclusion
We confirmed in our animal experiments that CVB3/MKP had cardiotoxicity. CVB3/MKP, CVB3/28, and CVB3/0 may share evolutionary convergence and the 5′untranslated region (5′UTR) may be associated with virulence phenotype. Our findings will provide a basis for identifying the genomic determinant of viral virulence of CVB3/MKP strain and phylogenetic relationship among different CVB3 strains.
doi:10.1186/1743-422X-11-33
PMCID: PMC3996064  PMID: 24555514
Coxsackievirus B3/MKP; Myocarditis; Phylogenetic analysis; Functional genomics
23.  Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain reaction detection of enterovirus RNA in clinical specimens. 
We describe a rapid method for extraction and detection of enterovirus RNA in clinical samples. By using magnetic bead technology, enterovirus RNA was efficiently and rapidly extracted from cerebrospinal fluid, stool, saliva, blood, pericardial fluid, urine, and cryopreserved or formalin-fixed solid tissue. Enterovirus RNA was then detected by reverse transcription followed by polymerase chain reaction amplification with primers designed to allow detection of most enterovirus serotypes. For detection of enteroviruses in specimens from patients with acute enteroviral disease, the overall sensitivity of enzymatic RNA amplification was greater than that of cell culture isolation, especially in blood specimens and in stool specimens from patients with acute cardiac disease. Enterovirus RNA was also detected in cryopreserved and archival formalin-fixed myocardial tissue from patients with acute myocarditis and chronic dilated cardiomyopathy. The ability to study archival specimens is of particular value in conducting retrospective investigation. The RNA extraction procedure used was considerably faster than extraction methods using organic reagents, used less hazardous reagents, and was of similar sensitivity. This detection protocol may therefore be useful both for the diagnosis of enterovirus infection and in studying the pathogenesis of acute and chronic enterovirus-induced disease.
Images
PMCID: PMC262616  PMID: 8380182
24.  The importance of cardiac MRI as a diagnostic tool in viral myocarditis-induced cardiomyopathy 
Netherlands Heart Journal  2009;17(12):481-486.
Myocarditis is an acute or chronic inflammatory disease of the myocardium which can be viral, postinfectious immune or primarily organ-specific autoimmune. Clinical manifestations of acute and chronic myocarditis are extremely varied, ranging from mild to severe. Affected patients may recover or develop (dilated) cardiomyopathy (DCM) with life-threatening symptoms including heart failure, conduction disturbances, arrhythmias, cardiogenic shock or sudden cardiac death.
The diagnosis of myocarditis is a challenging process and not only because of a diverse presentation; other problems are limited sensitivity of endomyocardial biopsies (EMB) and overlapping symptoms. Furthermore, the diagnosis is not well defined. However, early diagnosis is mandatory to address specific aetiology-directed therapeutic management in myocarditis that influences patient morbidity and mortality.
Currently, EMB remains the only way to confirm the presence of a viral genome and other histopathological findings allowing proper treatment to be implemented in cases of myocarditis. Increased recognition of the role of myocardial inflammatory changes has given rise to interest in noninvasive imaging as a diagnostic tool, especially cardiovascular magnetic resonance imaging (CMR). In this review we discuss the current role of CMR in the evaluation of myocarditis-induced inflammatory cardiomyopathies. (Neth Heart J 2009;17:481-6.)
PMCID: PMC2804081  PMID: 20087452
myocarditis; cardiac magnetic resonance imaging; cardiomyopathy
25.  Sequences specific for enterovirus detected in spinal cord from patients with motor neurone disease. 
BMJ : British Medical Journal  1994;308(6943):1541-1543.
OBJECTIVE--To investigate the association of enteroviruses with motor neurone disease, also known as amyotrophic lateral sclerosis. DESIGN--Analysis by enterovirus polymerase chain reaction of wax embedded material from spinal cords taken at necropsy from subjects with motor neurone disease and from age and sex matched controls. SETTING--Specimens were collected in the west of Scotland and in London between 1982 and 1992. RESULTS--Sequences specific for a non-poliovirus type enterovirus were detected in spinal cord tissue from subjects with motor neurone disease. Amplification of a 414 base RNA target sequence in the conserved enterovirus 5' untranslated region from wax embedded tissue sections was successful in tissue from eight of 11 cases of sporadic motor neurone disease, one of two cases of familial motor neurone disease, and the one case of poliomyelitis, but not in the six matched controls or one case of antecedent poliomyelitis. In addition, sequences were detected in spinal cords from one monkey infected with wild type poliovirus and one monkey infected with polio vaccine. Comparison of sequences from cases of motor neurone disease with sequences of corresponding regions of the 5' untranslated regions of known picornaviruses showed them to be tightly grouped within the enterovirus genus closely related to coxsackievirus type B but not to polioviruses. Sequences derived from different parts of the spinal cord of the same subjects were identical, but sequences differed between individual subjects. CONCLUSIONS--Conserved enteroviral sequences closely related to coxsackie B virus sequences were detectable in spinal cords from subjects with sporadic motor neurone disease and from one subject with possible familial motor neurone disease.
PMCID: PMC2540520  PMID: 8019310

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