Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

Systematic Review of PCR Proof of Parvovirus B19 Genomes in Endomyocardial Biopsies of Patients Presenting with Myocarditis or Dilated Cardiomyopathy.

BACKGROUND: Diverse viral infections have been associated with myocarditis (MC) and dilated cardiomyopathy (DCM). In this meta-analysis, we summarize the published results on the association of parvovirus B19 (B19V) genomes with human MC/DCM versus controls. METHODS: n = 197 publications referring to B19V and MC or DCM were retrieved using multiple PubMed search modes. Out of these, n = 29 publications met the inclusion criteria with data from prospective analyses on >10 unselected patients presenting with MC or DCM (dataset: MA01). Data retrieved simultaneously from both controls and MC/DCM patients were available from n = 8 from these publications (dataset: MA02). RESULTS: In the dataset MA01 B19V genomes were detected in 42.6% of the endomyocardial biopsies (EMB) in this cohort by PCR. In the dataset MA02 comprising n = 638 subjects, there was no statistically significant different rate of B19V positivity in myocardial tissues comparing controls (mean: 38.8 + 24.1%) versus the MC/DCM-patients (45.5 + 24.3%; p = 0.58). There was also no statistical difference between the positivity rate of B19V genomes in myocardial tissues of MA01 (46.0 + 19.5%) and the two patient groups of MA02 (p > 0.05). CONCLUSIONS: This systematic review reveals that the mean rate of PCR detected B19V genomes in patients presenting with MC/DCM does not differ significantly from the findings in control myocardial tissues. These data imply pathogenetically insignificant latency of B19V genomes in a proportion of myocardial tissues, both in MC-/DCM-patients and in controls. More information (i.e., replicative status, viral protein expression) is pertinent to achieve a comprehensive workup of myocardial B19V infection.

Enterovirus Persistence in Cardiac Cells of Patients With Idiopathic Dilated Cardiomyopathy Is Linked to 5' Terminal Genomic RNA-Deleted Viral Populations With Viral-Encoded Proteinase Activities.

BACKGROUND: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. METHODS: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. RESULTS: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. CONCLUSIONS: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

Parvovirus B19 in Endomyocardial Biopsy of Patients With Idiopathic Dilated Cardiomyopathy: Foe or Bystander?

BACKGROUND: Parvovirus B19 (PVB19) has emerged as one of the viruses possibly inducing chronic myocarditis and subsequent idiopathic dilated cardiomyopathy (IDCM). The aim of this work was to investigate the presence and long-term consequences of PVB19-DNA within myocardial biopsies from patients with IDCM and to compare the findings with those from donor hearts (control group). METHODS AND RESULTS: Forty hospitalized IDCM patients (age 47 ± 12 y) with mean left ventricular ejection fraction 27 ± 12% were included. The presence of PVB19-DNA in myocardial biopsies and of IgG and IgM antibodies in patient sera was analyzed. The control group consisted of 20 donor hearts. The follow-up time was 112 ± 57 months. PVB19-DNA was found in myocardial biopsies of both patients (73%) and control samples (55%; P = .25).Three deaths and 8 heart transplantations occurred in the IDCM group, and 6 deaths in the control group (ie, the recipients of the control hearts). No difference in transplantation-free survival between the PVB19-DNA positive/negative IDCM patients or transplant recipients was found. CONCLUSIONS: PVB19-DNA is a common finding in both patients with IDCM and in healthy donor hearts, not affecting prognosis. These findings support the view that PVB19 is an innocent bystander, frequently found in myocardium with low DNA copies, and not a plausible cause of IDCM.

First evidence of the presence of adenovirus type 8 in myocardium of patients with severe idiopathic dilated cardiomyopathy.

Previous studies have detected adenovirus and cytomegalovirus (CMV) in cardiac tissue of patients with myocarditis. Therefore, in this study, we investigated the frequency of these viruses, which may be involved in the development of severe dilated cardiomyopathy (DCM). Myocardial tissue from of 23 cardiac transplant candidates with acute idiopathic DCM below the age of 40 years were analyzed by amplification of adenovirus and CMV DNA and subsequent sequencing. Adenovirus was detected in four (17.4%) and CMV in one (4.3%) of the patients. All controls were negative for the presence of both viruses. Our study shows that myocardial infection with adenovirus may play an important role in the pathogenesis of severe DCM and suggests that vaccination against adenovirus might be helpful in decreasing the prevalence of severe idiopathic DCM. This is the first study in which adenovirus type 8 has been detected in the hearts of patients with DCM.

[Efficiency of immunosuppressive therapy in virus-negative and virus-positive patients with morphologically verified lymphocytic myocarditis]. / Éffektivnost' immunosupressivnoi terapii u virus-negativnykh i viruspozitivnykh bol'nykh s morfologicheski verifitsirovannym limfotsitarnym miokarditom.

AIM: To evaluate the efficiency of immunosuppressive therapy (IST) in virus-negative (V-) and virus-positive (V+) patients with lymphocytic myocarditis (LM). SUBJECTS AND METHODS: 60 patients (45 males) (mean age 46.7±11.8 years) with dilated cardiomyopathy (mean left ventricular (LV) end diastolic size (EDS) 6.7±0.7 cm; ejection fraction (EF) 26.2±9.1%) were examined. The diagnosis of active/borderline LM was verified by right ventricular endomyocardial biopsy in 38 patients, by intraoperative LV biopsy in 10, in the study of explanted hearts from 3 patients and at autopsy in 9. The investigators determined the genomes of parvovirus B19, herpes viruses types 1, 2 and 6, Epstein-Barr (EBV), zoster, and cytomegalovirus in the blood and myocardium and, if antibodies were present in the blood, hepatitis B and C viruses, as well as antibodies against antigens in the endothelium, cardiomyocytes and their nuclei, smooth muscles, fibers of the conducting system. IST was used in terms of histological, immune, and viral activities. IST was performed in 22 V+ patients (Group 1) and in 24 V- patients (Group 2); this was not done in 10 V+ patients (Group 3) and V- patients (Group 4). IST comprised methylprednisolone at a mean dose of 24 mg/day (n=40), hydroxychloroquine 200 mg/day (n=20), azathioprine at a mean dose of 150 mg/day (n=21); antiviral therapy included acyclovir, ganciclovir, intravenous immunoglobulin (n=24). The follow-up period was 19 (7.3-40.3) months. RESULTS: The viral genome was detected in the myocardium of 32 patients who made up a V+ group. The degree of histological activity did not differ in relation to the presence of viral genome in the myocardium. The degree of immune activity (anticardiolipin antibody titers) in the V+ patients was as high as that in V- ones. At baseline, the V+ patients had a significantly higher LV EDS and a lower EF than the V- patients. Overall, IST only could lead to a significant increase in EF (from 26.5±0.9 to 36.0±10.8%; p<0.001) and reductions in NYHA functional class from III to II (p<0.001), LV EDS (from 6.7±0.7 to 6.4±0.8 cm; p<0.01), pulmonary artery systolic pressure (from 48.9±15.5 to 39.4±11.5 mm Hg (p<0.01); the IST group had significantly lower mortality rates than the non-IST group (23.9 and 64.3%; p<0.01). At the same time, a significant trend was seen in both V- and V+ patients. The mortality rate in the V+ patients, as a whole, was higher (46.9 and 17.9%; p<0.05). CONCLUSION: IST leads to a significant improvement of functional indices and it is associated with lower mortality rates in both myocardial V- and V+ patients with LM. A more than 10% EF increase in the first 2 months is associated with a good prognosis. The presence of viral genome in the myocardium (primarily herpesviruses rather than parvovirus-19) is accompanied by more severe initial dysfunction, a less pronounced effect of IST, and higher mortality rates. However, the positive effect of IST also persists in V+ patients. No positive changes (a decrease in EF was observed) were absent only in IST-naïve V+ patients.

Enterovirus but not Parvovirus B19 is associated with idiopathic dilated cardiomyopathy and endomyocardial CD3, CD68, or HLA-DR expression.

We assessed Enterovirus (EV) &Parvovirus B19 (PVB19) genomes and CD3, CD68&HLA-DR detection in dilated cardiomyopathies (DCM). EV&PVB19 genomes and CD3, CD68&HLA-DR were detected by PCR and immunohistochemistry assays in 115 endomyocardial biopsies obtained in 13 idiopathic DCM (iDCM) and 10 explained DCM (eDCM) patients. Results were compared with those of 47 atrial surgical samples (47 surgery controls) and 22 autoptic cardiac samples (11 healthy heart controls) (2008-2014, Reims, France). EV was detected in 23.1% of iDCM patients but not in eDCM and controls (P = 0.003) (viral load 803 copies/µg). PVB19 was detected in 76.9%, 80.0%, 63.6% and 78.2% of iDCM, eDCM, healthy heart and surgery controls (P = 0.99) with a mean viral load of 413, 346, 1,428, and 71 copies/µg. CD3, CD68 or HLA-DR were detected in 100 and 50% of EV and PVB19 "mono-infected" iDCM patients. EV was exclusively detected in iDCM cases in association with CD3, CD68, or HLA-DR indicating that EV could be an etiological cause in a subset of iDCM cases. By contrast the equal frequent detection of PVB19 in iDCM cases and controls without association with CD3, CD68, or HLA-DR suggested that PVB19 could be a bystander in many DCM cases. J. Med. Virol. 89:55-63, 2017. © 2016 Wiley Periodicals, Inc.

A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19.

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.

Relevance of cardiac parvovirus B19 in myocarditis and dilated cardiomyopathy: review of the literature.

Over the last decade, parvovirus B19 (B19V) has frequently been linked to the pathogenesis of myocarditis (MC) and its progression towards dilated cardiomyopathy (DCM). The exact role of the presence of B19V and its load remains controversial, as this virus is also found in the heart of healthy subjects. Moreover, the prognostic relevance of B19V prevalence in endomyocardial biopsies still remains unclear. As a result, it is unclear whether the presence of B19V should be treated. This review provides an overview of recent literature investigating the presence of B19V and its pathophysiological relevance in MC and DCM, as well as in normal hearts. In brief, no difference in B19V prevalence is observed between MC/DCM and healthy control hearts. Therefore, the question remains open whether and how cardiac B19V may be of pathogenetic importance. Findings suggest that B19V is aetiologically relevant either in the presence of other cardiotropic viruses, or when B19V load is high and/or actively replicating, which both may maintain myocardial (low-grade) inflammation. Therefore, future studies should focus on the prognostic relevance of the viral load, replicative status and virus co-infections. In addition, the immunogenetic background of MC/DCM patients that makes them susceptible to develop heart failure upon presence of B19V should be more thoroughly investigated.

Viral myocarditis.

PURPOSE OF REVIEW: The article traces the pathways leading from viral infection of the heart by coxsackievirus B3 to autoimmune myocarditis in its various manifestations. RECENT FINDINGS: Myocarditis can be induced by a number of different infectious agents and represents a significant cause of death especially in young individuals. Following infection, patients may develop lymphocytic, eosinophilic, or giant cell/granulomatous myocardial inflammation. It can lead to infectious dilated cardiomyopathy, a disease frequently requiring cardiac transplantation. Although acute viral myocarditis is frequently subclinical and recovery may be spontaneous, treatment of chronic myocarditis is currently unsatisfactory. Ongoing disease may be because of persistent virus in the heart or to immunopathic attack. Depending on the cause, treatment may be antiviral or immunosuppressive. Endomyocardial biopsy is proving of value in determining cause and deciding future therapy. A great deal of information about the pathogenesis of myocarditis has been gained from experimental models in rodents using heart disease induced by infection using coxsackievirus B3 or by immunization with cardiac myosin. SUMMARY: Treatment of myocarditis is still problematic and may depend on etiologic diagnosis to distinguish infectious from immune-mediated disease. Both pathogenic mechanisms may co-occur in individual patients. In the future, treatment may depend upon endomyocardial biopsy, immunohistologic testing, improved imaging, and molecular genetic analysis for providing more precise diagnoses.

The Impact of Specific Viruses on Clinical Outcome in Children Presenting with Acute Heart Failure.

The presence and type of viral genomes have been suggested as the main etiology for inflammatory dilated cardiomyopathy. Information on the clinical implication of this finding in a large population of children is lacking. We evaluated the prevalence, type, and clinical impact of specific viral genomes in endomyocardial biopsies (EMB) collected between 2001 and 2013 among 63 children admitted to our hospital for acute heart failure (median age 2.8 years). Viral genome was searched by polymerase chain reaction (PCR). Patients underwent a complete two-dimensional echocardiographic examination at hospital admission and at discharge and were followed-up for 10 years. Twenty-seven adverse events (7 deaths and 20 cardiac transplantations) occurred during the follow-up. Viral genome was amplified in 19/63 biopsies (35%); PVB19 was the most commonly isolated virus. Presence of specific viral genome was associated with a significant recovery in ejection fraction, compared to patients without viral evidence (p < 0.05). In Cox-regression analysis, higher survival rate was related to virus-positive biopsies (p < 0.05). When comparing long-term prognosis among different viral groups, a trend towards better prognosis was observed in the presence of isolated Parvovirus B19 (PVB19) (p = 0.07). In our series, presence of a virus-positive EMB (mainly PVB19) was associated with improvement over time in cardiac function and better long-term prognosis.

Viral Nucleic Acids in the Serum Are Dependent on Blood Sampling Site in Patients with Clinical Suspicion of Myocarditis.

BACKGROUND: The meaning of viral nucleic acids in the myocardium in many cases is difficult for clinical interpretation, whereas the presence of viral nucleic acids in the serum is a marker of active infection. We determined the diagnostic value of viral nucleic acids in ventricular serum and peripheral serum samples in comparison with endomyocardial biopsy (EMB) specimens in patients with clinically suspected myocarditis. METHODS: The viral nucleic acid evaluation was performed in serum samples and EMB specimens by real-time PCR in 70 patients (age: 47 ± 16 years). The biopsy specimens were examined by histo- and immunohistochemistry to detect inflammatory response. RESULTS: The viral nucleic acids were detected in ventricular and peripheral serum, and EMB samples of 10 (14%), 14 (20%), and 32 (46%) patients, respectively. Notably, viral nucleic acids of the same virus as in the EMB sample were present more often in ventricular than in peripheral serum (60 vs. 7%, p = 0.01). A significant concurrence was observed between the positive and the negative results of viral nucleic acids present in EMB and ventricular serum (p = 0.0001). CONCLUSIONS: The detection of the same viral nucleic acid type in the myocardium and in ventricular serum being significantly more frequent than in the peripheral serum may suggest that the site of the blood collection is important for more precise and reliable confirmation of the active viral replication in the heart.

Serological and Molecular Biological Studies of Parvovirus B19, Coxsackie B Viruses, and Adenoviruses as Potential Cardiotropic Viruses in Bulgaria.

BACKGROUND: Inflammatory diseases of the heart (myocarditis, pericarditis) are commonly caused by viruses. Among the human cardiotropic viruses, parvovirus B19, Coxsackie B viruses, and adenoviruses play a leading role. AIM: The aim of the present study was to determine the presumptive causative role of parvovirus B19, Coxsackie B viruses, and adenoviruses in the development of myocarditis, pericarditis and dilated cardiomyopathy by demonstrating the presence of specific antiviral antibodies or viral DNA in patients' serum samples. MATERIALS AND METHODS: We tested serum samples collected between 2010 and 2014 from 235 patients with myocarditis (n=108), pericarditis (n=79), myopericarditis (n=19), dilated cardiomyopathy (n=7), and fever of unknown origin accompanied by cardiac complaints (n=22). The mean age of patients with the standard deviation was 33 ± 18 years. Serological and molecular methods (ELISA for specific IgM/IgG antibodies to parvovirus B19 and IgM antibodies to Coxsackie B viruses and adenoviruses, and PCR for detection of parvovirus B19 in serum samples, respectively) were used in the study. RESULTS: Of all tested 235 serum samples, in 60 (25.5%) positive results for at least one of the three tested viruses were detected. Forty out of these 235 serum samples (17%) were Coxsackie B virus IgM positive. They were found in 17% (18/108) of the patients with myocarditis, in 15% (12/79) of those with pericarditis, in 16% (3/19) of those with myopericarditis and in 32% (7/22) in those with fever of unknown origin. The 63 Coxsackie B virus IgM negative patient's serum samples were tested by ELISA for presence of adenovirus IgM antibodies. Such were found in 4 patients with pericarditis and in 2 patients with fever of unknown origin. Every IgM negative sample (n=189) for Coxsackie B and adenovirus was further tested by ELISA for parvovirus B19 IgM/IgG antibodies. B19-IgM antibodies were detected in 14 patients (7.4%). The percentages for B19-IgM antibodies was 8% (7/90), 5% (3/63) and 31% (4/13) in the patients affected with myocarditis, pericarditis, and fever of unknown origin, respectively. Protective B19-IgG antibodies were found in 108 (57%) of the samples. A B19-PCR signal was detected in all the patients who were B19-IgM positive, and in only 1 patient with positive B19-IgG result, the latter presenting with dilated cardiomyopathy. CONCLUSION: The present study shows the involvement of Coxsackie B, parvovirus B19 and adenoviruses in the development of inflammatory diseases of the heart (myocarditis and pericarditis). It is the first ever study in the country that simultaneously analyzes the prevalence of the three major human cardiotropic viruses.

Role of coxsackievirus and adenovirus receptor (CAR) expression and viral load of adenovirus and enterovirus in patients with dilated cardiomyopathy.

Enteroviruses (EVs) and adenoviruses (AdVs) are two important etiological agents of viral myocarditis and dilated cardiomyopathy (DCM). Both these viruses share a common receptor, the coxsackievirus and adenovirus receptor (CAR), for their infection. However, the role of viral load and CAR expression in disease severity has not yet been completely elucidated. The present study aimed to determine viral load of EV and AdV in DCM patients and correlate them with the level of CAR expression in these patients. Sixty-three DCM cases and 30 controls, each of whom died of heart disease other than DCM and non-cardiac disease respectively, were included. Viral load was determined by TaqMan real-time PCR using primers and probes specific for the AdV hexon gene and the 5'UTR region of EV. The CAR mRNA level was semi-quantitated by RT-PCR, and antigen expression was studied by immunohistochemistry. A significantly high AdV load (p < 0.05) and CAR expression (p < 0.05) were observed in DCM cases versus controls, whereas the EV load showed no significant difference. The data suggests a clinical threshold of 128 AdV copies/500 ng of DNA for DCM, with 66.7 % sensitivity and 65 % specificity. A positive correlation between AdV load and CAR expression (p < 0.001) was also observed in DCM cases. The high adenoviral load and increased CAR expression in DCM and their association with adverse disease outcome indicates role of both virus and receptor in disease pathogenesis. Thus, the need for targeting both the virus and the receptor for treatment of viral myocarditis and early DCM requires further confirmation with larger studies.

Clinical Significance of Viral Genome Persistence in the Myocardium of Patients with Dilated Cardiomyopathy.

BACKGROUND: The impact of myocardial viral persistence on the clinical outcome of patients with dilated cardiomyopathy (DCM) is still open to question. METHODS: Fifty-two patients with DCM were enrolled and followed for a median of 3.8 years with respect to death or heart transplantation. Studied patients were clinically stable for at least 6 months before hospitalization. They underwent coronary angiography and endomyocardial biopsy. Specimens were examined by histo- and immunohistochemistry, and the viral genomes of parvovirus B19, cytomegalovirus (CMV), Coxsackie B virus (CVB), and hepatitis B and C viruses were studied by real-time polymerase chain reaction. RESULTS: Forty-two out of 52 patients were available for clinical follow-up. The viral genome was detected in the myocardium of 32 out of 42 patients. Among the viruses studied, CMV and CVB were the most frequently found. Nine out of 42 patients achieved the predefined study end point. No statistically significant correlation was found between the presence of a persistent viral genome and study end point. No statistically significant relationship between viral genomes studied and immunohistology results was detected. CONCLUSIONS: High prevalence of a viral genome in the myocardium of patients with DCM did not have an influence on their long-term clinical outcome.

Coxsackievirus B detection in cases of myocarditis, myopericarditis, pericarditis and dilated cardiomyopathy in hospitalized patients.

Coxsackieviruses B (CV­B) 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 CV­B 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 (RT­PCR) 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. CV­B was detected in 28 of the 102 patients. The sequence analysis demonstrated that 27 strains were identical to CV­B3 and only one strain was identical to CV­B1. Furthermore, VP1 in the heart biopsies was detected in enterovirus­positive cases, as revealed by RT­PCR. Pericarditis infection was more frequent than myocarditis (P<0.05) or myopericarditis (P=0.05). The epidemiological data demonstrate that CV­B 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 CV­B may significantly contribute to heart infections.

Ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy.

BACKGROUND: Common causative agents in the development of inflammatory cardiomyopathy include cardiotropic viruses such as coxsackievirus B3 (CVB3). Here, we investigated the role of the ubiquitin-like modifier interferon-stimulated gene of 15 kDa (ISG15) in the pathogenesis of viral cardiomyopathy. METHODS AND RESULTS: In CVB3-infected mice, the absence of protein modification with ISG15 was accompanied by a profound exacerbation of myocarditis and by a significant increase in mortality and heart failure. We found that ISG15 in cardiomyocytes contributed significantly to the suppression of viral replication. In the absence of an intact ISG15 system, virus titers were markedly elevated by postinfection day 8, and viral RNA persisted in ISG15(-/-) mice at postinfection day 28. Ablation of the ISG15 protein modification system in CVB3 infection predisposed mice to long-term disease with deposition of collagen fibers, all leading to inflammatory cardiomyopathy. We found that ISG15 acts as part of the intrinsic immunity in cardiomyocytes and detected no significant effects of ISG15 modification on the cellular immune response. ISG15 modification of CVB3 2A protease counterbalanced CVB3-induced cleavage of the host cell eukaryotic initiation factor of translation eIF4G in cardiomyocytes, thereby counterbalancing the shutoff of host cell translation in CVB3 infection. We demonstrate that ISG15 suppressed infectious virus yield in human cardiac myocytes and the induction of ISG15 in patients with viral cardiomyopathy. CONCLUSIONS: The ISG15 conjugation system represents a critical innate response mechanism in cardiomyocytes to fight the battle against invading pathogens, limiting inflammatory cardiomyopathy, heart failure, and death. Interference with the ISG15 system might be a novel therapeutic approach in viral cardiomyopathy.

Correlations among persistent viral infection, heart function and Chinese medicine syndromes in dilated cardiomyopathy patients.

OBJECTIVE: To investigate the correlations among persistent viral infection, heart function and Chinese medicine (CM) difined-syndromes in patients with dilated cardiomyopathy (DCM). METHODS: Fifty patients with DCM in the First Affiliated Hospital of Zhejiang Chinese Medical University from October 2009 to December 2011 were selected as the research subjects, and 30 healthy people were simultaneously selected as the normal control group to detect persistent viral infections after admission. The CM syndrome type and grade of heart function were then evaluated. The expression level of Coxsackie adenovirus receptor (CAR) was detected using the flow cytometry (FCM) technique, coxsackie virus RNA (CVB-RNA) using reverse transcription polymerase chain reaction (RTPCR), and the plasma brain natriuretic peptide (BNP) level with a Triage meter plus diagnosis instrument. Finally, the parameters such as left ventricular end diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) were measured by ultrasonic cardiogram. Person correlation analysis was used for measured data, Spearman correlation analysis for rating data, and the Chi-square test for numerical data. RESULTS: CVB-RNA was positive in 22 patients (44%) with DCM, while only 6 cases (20%) were CVB-RNA-positive in the normal control group, with a significant difference between the two groups (P<0.01). The expression level of CAR was significantly elevated in the DCM group compared with the normal control group (P<0.01). In CVB-RNA-positive patients (22 cases), the expression level of CAR was significantly higher than in CVB-RNA-negative patients (28 cases; P<0.01). In the DCM patients, there was a positive correlation between the CAR expression and the BNP level (r=0.34, P<0.05), while no significant difference was found between the CAR expression and the LVEF and LVEDd (r=-0.32, 0.30, P>0.05). There was no clear correlation between virus infection and the CM syndrome types in DCM patients (r=-0.22, P>0.05). According to the sequence of syndrome types: phlegm → qi deficiency → blood stasis → hydroretention with asthenic yang (from low to high), a positive correlation was existed between the BNP levels and CM syndrome types (r=0.139, P<0.05). CONCLUSION: The expression of CAR on the surface of white cells could be used to detect persistent viral infection. The expression level of CAR and heart function in DCM patients were highly correlated. The expression level of BNP may serve as an objective index for differentiating CM syndromes for patients with DCM.

Ectopic trypsin in the myocardium promotes dilated cardiomyopathy after influenza A virus infection.

We have previously reported that ectopic trypsin in the myocardium triggers acute myocarditis after influenza A virus (IAV) infection. As myocarditis is a common precursor to dilated cardiomyopathy (DCM), the aim of the present study was to investigate the influence of trypsin on the progression of DCM after IAV infection. IAV-infected mice treated with saline or trypsin inhibitor were euthanized on days 0, 9, 20, 40 and 60 postinfection. Trypsin expression colocalized with myocardial inflammatory loci and IAV-induced myocarditis peaked on day 9 postinfection and alleviated by day 20 but persisted until day 60 postinfection, even though replication of IAV was not detected from day 20 postinfection. Similar time courses were observed for the activation of pro-matrix metalloproteinase (pro-MMP)-9 and expression of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α. Degradation of collagen type I, proliferation of ventricular interstitial collagen, and expression of collagen type I and III mRNA increased significantly during acute and chronic phases; collagen type III mRNA increased more significantly than collagen type I mRNA. Cardiac function progressively deteriorated with progressive left ventricular dilation. The trypsin inhibitor aprotinin suppressed pro-MMP-9 activation and cytokine release, alleviated myocardial inflammation, and restored collagen metabolism during acute and chronic phases of myocarditis. This effectively prevented ventricular dilation and improved cardiac function. These results suggest that ectopic trypsin in the myocardium promoted DCM through chronic activation of pro-MMP-9, persistent induction of cytokines, and mediation of collagen remodeling. Pharmacological inhibition of trypsin activity might be a promising approach for the prevention of viral cardiomyopathy.