Myocarditis and inflammatory cardiomyopathy: current evidence and future directions.

Inflammatory cardiomyopathy, characterized by inflammatory cell infiltration into the myocardium and a high risk of deteriorating cardiac function, has a heterogeneous aetiology. Inflammatory cardiomyopathy is predominantly mediated by viral infection, but can also be induced by bacterial, protozoal or fungal infections as well as a wide variety of toxic substances and drugs and systemic immune-mediated diseases. Despite extensive research, inflammatory cardiomyopathy complicated by left ventricular dysfunction, heart failure or arrhythmia is associated with a poor prognosis. At present, the reason why some patients recover without residual myocardial injury whereas others develop dilated cardiomyopathy is unclear. The relative roles of the pathogen, host genomics and environmental factors in disease progression and healing are still under discussion, including which viruses are active inducers and which are only bystanders. As a consequence, treatment strategies are not well established. In this Review, we summarize and evaluate the available evidence on the pathogenesis, diagnosis and treatment of myocarditis and inflammatory cardiomyopathy, with a special focus on virus-induced and virus-associated myocarditis. Furthermore, we identify knowledge gaps, appraise the available experimental models and propose future directions for the field. The current knowledge and open questions regarding the cardiovascular effects associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are also discussed. This Review is the result of scientific cooperation of members of the Heart Failure Association of the ESC, the Heart Failure Society of America and the Japanese Heart Failure Society.

Acute flaccid paralysis due to Echovirus 30 in an immunosuppressed transplant recipient.

An Italian 13-year-old boy immunosuppressed due to kidney transplant presented in November 2018 with acute flaccid paralysis with anterior horn cell involvement resembling the clinical, radiological, and laboratory features of poliomyelitis. Enterovirus was molecularly identified in cerebral spinal fluid and stool samples and the sequence analysis of the VP1 gene of enterovirus genome revealed the presence of Echovirus 30 both in CSF and in stool samples. Echovirus 30 is an emerging neurotropic virus able to cause outbreaks of aseptic meningitis and meningoencephalitis all over the world, but acute flaccid paralysis is not a classical manifestation. A 6-month follow-up revealed a poor outcome with severe motor deficits and only slight improvement in disability. Clinicians must be aware of the possible role of Echovirus 30 in acute flaccid paralysis and active surveillance should consider the possible influence of immunosuppression on the symptoms caused by the widening spectrum of enterovirus infections.

Distinct migratory pattern of naive and effector T cells through the blood-CSF barrier following Echovirus 30 infection.

BACKGROUND: Echovirus 30 (E-30) is one of the most frequently isolated pathogens in aseptic meningitis worldwide. To gain access to the central nervous system (CNS), E-30 and immune cells have to cross one of the two main barriers of the CNS, the epithelial blood-cerebrospinal fluid barrier (BCSFB) or the endothelial blood-brain barrier (BBB). In an in vitro model of the BCSFB, it has been shown that E-30 can infect human immortalized brain choroid plexus papilloma (HIBCPP) cells. METHODS: In this study we investigated the migration of different T cell subpopulations, naive and effector T cells, through HIBCPP cells during E-30 infection. Effects of E-30 infection and the migration process were evaluated via immunofluorescence and flow cytometry analysis, as well as transepithelial resistance and dextran flux measurement. RESULTS: Th1 effector cells and enterovirus-specific effector T cells migrated through HIBCPP cells more efficiently than naive CD4+ T cells following E-30 infection of HIBCPP cells. Among the different naive T cell populations, CD8+ T cells crossed the E-30-infected HIBCPP cell layer in a significantly higher number than CD4+ T cells. A large amount of effector T cells also remained attached to the basolateral side of the HIBCPP cells compared with naive T cells. Analysis of HIBCPP barrier function showed significant alteration after E-30 infection and trans- as well as paracellular migration of T cells independent of the respective subpopulation. Morphologic analysis of migrating T cells revealed that a polarized phenotype was induced by the chemokine CXCL12, but reversed to a round phenotype after E-30 infection. Further characterization of migrating Th1 effector cells revealed a downregulation of surface adhesion proteins such as LFA-1 PSGL-1, CD44, and CD49d. CONCLUSION: Taken together these results suggest that naive CD8+ and Th1 effector cells are highly efficient to migrate through the BCSFB in an inflammatory environment. The T cell phenotype is modified during the migration process through HIBCPP cells.

The neonatal Fc receptor is a pan-echovirus receptor.

Echoviruses are amongst the most common causative agents of aseptic meningitis worldwide and are particularly devastating in the neonatal population, where they are associated with severe hepatitis, neurological disease, including meningitis and encephalitis, and even death. Here, we identify the neonatal Fc receptor (FcRn) as a pan-echovirus receptor. We show that loss of expression of FcRn or its binding partner beta 2 microglobulin (ß2M) renders cells resistant to infection by a panel of echoviruses at the stage of virus attachment, and that a blocking antibody to ß2M inhibits echovirus infection in cell lines and in primary human intestinal epithelial cells. We also show that expression of human, but not mouse, FcRn renders nonpermissive human and mouse cells sensitive to echovirus infection and that the extracellular domain of human FcRn directly binds echovirus particles and neutralizes infection. Lastly, we show that neonatal mice expressing human FcRn are more susceptible to echovirus infection by the enteral route. Our findings thus identify FcRn as a pan-echovirus receptor, which may explain the enhanced susceptibility of neonates to echovirus infections.

Seroepidemiology of echovirus 30 in Korean children.

BACKGROUND: Although aseptic meningitis associated with echovirus type 30 has emerged as a global public health concern, no data have been reported on Children's immune status against echovirus type 30. The current study aimed to investigate the seropositivity among Korean children for antibodies against echovirus 30. METHODS: Two hundred and fifty residual serum samples were collected at St. Paul's Hospital. Individuals were categorized by age into four groups: group 1 (3 months-2 years), group 2 (3-6 years), group 3 (7-10 years) and group 4 (11-15 years). Neutralizing antibodies against echovirus 30 were measured. RESULTS: Seroprotective neutralizing antibodies against echovirus 30 were detected in 129 (49%) individuals. Seropositivity rates were 23%, 48%, 55% and 73% in groups 1-4, respectively. For antibody titers, 1:256-1:512 was the highest neutralizing antibody titer range in group 2, while 1:1024-1:2048 in group 3 and 4. Among the seropositive individuals in group 3 and 4, 6% and 12% had neutralizing antibody titers of 1:2048, respectively. CONCLUSIONS: The seropositivity rate increased significantly with age. The distribution of neutralizing antibody titers varied by age group, and higher ranges of neutralizing antibody titers were observed in higher age groups. These findings suggest high susceptibility to echovirus 30 infection in children younger than 2 years old. Echovirus 30 infection in childhood may have contributed to increased neutralizing antibody titers with age.

Lower anti-echovirus antibody responses in children presenting to hospital with asthma exacerbations.

BACKGROUND: Rhinoviruses from the Enterovirus genus cause frequent infections and induce remarkably high titres of anticapsid antigen antibodies in asthmatics, while the prevalence of neutralising antibodies to the gut-trophic echoviruses from the same genus is diminished. OBJECTIVE: To assess the absolute and specific antibody titres to VP1 antigens of the gut-trophic enteroviruses, echovirus 30 and Sabin 1 poliovirus, in asthmatic and non-asthmatic children. METHODS: Recombinant polypeptides representing the VP1 capsid antigens of echovirus 30 and Sabin poliovirus 1 were produced. Their ability to bind IgG1 antibodies from the plasma of asthmatic (n = 45) and non-asthmatic (n = 29) children were quantitated by immunoassays that incorporated immunoabsorptions to remove cross-reactivity. RESULTS: The IgG1 antibody titres and prevalence of antibody binding to echovirus 30 were significantly lower for asthmatic children compared to controls (P < 0.05) and inversely correlated with total IgE levels for the whole study population (r = -0.262; P < 0.05). There was no difference in the prevalence and titre between groups to the VP1 antigen of Sabin poliovirus. Anti-tetanus toxoid titres measured for comparison did not correlate with anti-echovirus or poliovirus, but correlated with anti-rhinovirus titres in controls but not asthmatics, where the titres were higher for the asthmatic group. CONCLUSIONS AND CLINICAL RELEVANCE: The associations of lower antibody titres of asthmatic children to echovirus reported here and those of our previous findings of a heightened response to rhinovirus suggest a dichotomy where respiratory enterovirus infection/immunity increases the probability of developing asthma and enteric infections lower the risk. This provides further support for the concept of intestinal infection playing a key role in the development of allergic respiratory disease.

Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections.

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus.

Expression of innate immunity genes and damage of primary human pancreatic islets by epidemic strains of Echovirus: implication for post-virus islet autoimmunity.

Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.

Molecular epidemiology and dual serotype specificity detection of echovirus 11 strains in Finland.

Echovirus 11 (E-11) has been one of the most frequently discovered human enterovirus (HEV) in Finland during the past few years. We have studied molecular epidemiological patterns of E-11 from 1993 to 2007 exploiting the 257-nucleotide region in the 5'-part of the VP1 used for genetic typing of HEV. Designated genogroup D strains had a striking prevalence among the Finnish strains, a finding in accordance with the recent data from other geographical regions. The subgroup D4, harboring the oldest strains, had become extinct in the beginning of the millennium and D5 strains had taken over. Similarly, a new subgroup of D5 had started to diverge from the main D5 in 2006. However, in addition to endemic D strains, few single strains clustered also to genogroups A and C suggesting importation from more distant locations. The relatively large amino acid sequence variability between and within the genogroups favored the idea of antigenic differences. Neutralization assays confirmed that antigenic differences existed, although all studied E-11 strains were neutralized with antisera against the prototype strain Gregory. Five of the six studied strains belonging to genogroup D were, unexpectedly, also neutralized with antisera against coxsackievirus A9 Griggs.

An enterovirus strain isolated from diabetic child belongs to a genetic subcluster of echovirus 11, but is also neutralised with monotypic antisera to coxsackievirus A9.

An enterovirus strain (designated D207) isolated from a Slovakian diabetic child and originally serotyped as coxsackievirus A9 (CAV-9) was found to cause rapid cytolysis coinciding with severe functional damage of the surviving cells in primary cultures of human pancreatic islets. This finding prompted us to clone the isolate for full-length genome sequencing and molecular characterization as the prototype strain of CAV-9 is known to cause only minimal damage to insulin-producing beta-cells. Based on capsid-coding sequence comparisons, the isolate turned out to be echovirus 11 (E-11). Phylogenetic analyses demonstrated that E-11/D207 was closely related to a specific subgroup B of E-11 strains known to cause uveitis. To study further antigenic properties of isolate E-11/D207 and uveitis-causing E-11 strains, neutralization experiments were carried out with CAV-9- and E-11-specific antisera. Unlike the prototype strains, the isolate E-11/D207 and uveitis-causing E-11 strains were well neutralized with both CAV-9- and E-11-specific antisera. Attempts to identify recombination of the capsid coding sequences as a reason for double-reactivity using the Simplot analysis failed to reveal major transferred motifs. However, peptide scanning technique was able to identify antigenic regions of capsid proteins of E-11/D207 as well as regions cross-reacting with an antiserum raised to CAV-9. Thus, double specificity of E-11/D207 seems to be a real characteristic shared by the phylogenetically closely related virus strains in the genetic subgroup B of E-11.

Genetic and phenotypic diversity of echovirus 30 strains and pathogenesis of type 1 diabetes.

Several enterovirus serotypes should be considered as potentially diabetogenic. The capacity of an enterovirus to kill or impair the functions of human beta-cells can vary among the strains within a given serotype as shown previously for echovirus 9 and 30 (E-30). The evolution of E-30 has also shown patterns correlating with the global increase of type 1 diabetes incidence. In the present study, antigenic properties of a set of E-30 isolates were investigated and the results correlated with the previously documented beta-cell destructive phenotype of the strains, or to genetic clustering of the strains. No simple correlation between the three properties was observed. A full-length infectious clone was constructed and sequenced from one of the isolates found to be most destructive to beta-cells (E-30/14916net87). Phylogenetic analyses demonstrated that this strain was closely related to the E-30 prototype strain at the capsid coding region while outside the capsid region prototype strains of several other human enterovirus B serotypes clustered more closely. This suggests that the relatively greater pathogenicity of the strain might be based on properties of the genome outside of the structural protein coding region. Neutralizing antibody assays on sera from 100 type 1 diabetic patients and 100 controls using three different E-30 strains did not reveal differences between the groups. This finding does not support a previous proposition of aberrant antibody responses to E-30 in diabetic patients. It is concluded that identification of the genetic counterparts of pathogenicity of E-30 strains requires further studies.

Cerebrospinal fluid cytokines in enterovirus 71 brain stem encephalitis and echovirus meningitis infections of varying severity.

Taiwan has experienced several outbreaks of enterovirus 71 (EV71) infections since 1998. This study examined the quantitative relationship between specific cytokines in the cerebrospinal fluid (CSF) and the severity of EV71 brain stem encephalitis (BE), and investigated whether the CSF cytokine response differed from that to uncomplicated echovirus meningitis (EM). The study included 57 children with EV71 BE, of whom 24 had isolated BE, 24 had autonomic nervous system (ANS) dysregulation, and nine had pulmonary oedema (PE), and 15 children with EM. All were confirmed by virus culture. Mean CSF glucose, total protein and lactate levels were increased significantly in association with the severity of EV71 BE. The mean CSF concentration of interleukin (IL)-1beta in children with EV71 PE was significantly higher than in those with isolated BE. IL-6 and interferon (IFN)-gamma levels were significantly higher for EV71 PE and ANS dysregulation than for isolated BE. In contrast, EM was associated with high levels of IL-1beta and low levels of IFN-gamma. Cytokines in the central nervous system, as well as in blood, appear to be involved in the pathogenesis of EV71 BE.

Echovirus infection causes rapid loss-of-function and cell death in human dendritic cells.

Coxsackie B viruses (CVB) and Echoviruses (EV) form a single species; Human enterovirus B (HeV-B), within the genus Enterovirus. Although HeV-B infections are usually mild or asymptomatic, they can cause serious acute illnesses. In addition, HeV-B infections have been associated with chronic immune disorders, such as type 1 diabetes mellitus and chronic myocarditis/dilated cardiomyopathy. It has therefore been suggested that these viruses may trigger an autoimmune process. Here, we demonstrate that human dendritic cells (DCs), which play an essential role in orchestration of the immune response, are productively infected by EV, but not CVB strains, in vitro. Infection does not result in DC activation or the induction of antiviral immune responses. Instead, EV infection rapidly impedes Toll-like receptor-mediated production of cytokines and upregulation of maturation markers, and ultimately causes loss of DC viability. These results describe for the first time the effect of EV on the function and viability of human DCs and suggest that infection of DCs in vivo can impede regulation of immune responses.

[Molecular identification and genetic analysis of an echovirus 19-associated epidemic of aseptic encephalitis].

OBJECTIVE: To ascertain the pathogen of aseptic encephalitis epidemic in Long-Yan city in Fujian, and to find out the genetic characteristics of the virus. METHODS: Rapid detection of enteroviral RNA by reverse transcription polymerasechain reaction (RT-PCR) was directly carried out in cerebrospinal fluid(CSF) to isolate and identify the viruses from CSF at the same time, and to detect the neutralization antibody in two serum specimens collected in acute and convalescence phase. Nucleotides of VP1 region was also analyzed by constructing phylogenetic tree. RESULTS: ECHO 19 infection was rapidly diagnosed and sequence analysed by RT-PCR, and then echovirus type 19 from 16 of 30 CSF samples (53.33%) was isolated and detected using RD and Hep-2 cells simultaneity. The titer of ECHO 19 neutralization antibody became positive or increased by 4 times from acute to convalescence phase in 4 of the 5 patients. Phylogenetic analyses of the VP1 genes of these isolates showed that their nucleotides identity were 98.9% -100.0% which were different from those ECHO 19 from GeneBank database by 13.0%-22.4%. CONCLUSION: The etiology of the epidemic of aseptic encephalitis was attributed to ECHO 19. The method of molecular identification not only provided rapid diagnosis of enterovirus infections, but also information about the genetic character of the viruses.

Type 1 diabetes islet associated antibodies in subjects infected by echovirus 16.

AIMS/HYPOTHESIS: To determine whether the emergent infection by echovirus 16 that occurred in Cuba during the year 2000 was related to the presence of Type 1 diabetes associated autoantibodies. METHODS: The presence of ICA, IAA, GADA, IA2 antibodies and neutralizing antibodies (NtAb) to echovirus 16 were determined in sera from 38 infected children and adolescents and 80 control subjects, matched in sex, age, local residence and time of sample collection. RESULTS: The occurrence of a large-scale echovirus 16 epidemic was associated with the appearance of humoral autoimmune markers of Type 1 diabetes, especially for ICA, IAA and GADA. In the convalescent stage, ICA, IAA and GADA seroconversion was shown in 92.1%, 44.7% and 28.9% of echovirus 16 infected subjects. None of the 80 uninfected subjects had ICA or IAA, while one was GADA positive. ICA, IAA and GADA frequency was higher in the convalescent than in the acute stage (p<0.0005). A strong positive correlation was found between the NtAb to echovirus 16 and ICA titres in both acute and convalescent stage (r=0.91; p<0.0001, r=0.55; p=0.0003 respectively). CONCLUSION/INTERPRETATION: This work provides evidence of an association between echovirus 16 infection and the presence of Type 1 diabetes related antibodies (ICA, IAA and GADA). Our data show that the echovirus 16 infection might be capable of inducing a process of autoimmune beta-cell damage and support the hypothesis that enterovirus infections are important risk factors for the development of Type 1 diabetes.

[Intrathecal synthesis pattern of immunoglobulins in meningoencephalitis epidemic due to echovirus 9]. / Patrones de síntesis intratecal de inmunoglobulinas en epidemia de meningoencefalitis por echovirus 9.

INTRODUCTION: Epidemics of meningoencephalitis due to echovirus 9 were commonly occurred when a children population become susceptible for the first time in front the virus. OBJECTIVE: To present the intrathecal synthesis pattern of immunoglobulins of the epidemic that affected Cuba in 1999 and to probe the usefulness of reibergram and antibody index in the diagnostic and characterization of the outbreak. PATIENTS AND METHODS: 23 pediatric patients suffering from viral meningoencephalitis due to echovirus 9 were studied in the income moment. Serum and cerebrospinal fluid IgA, IgM, IgG, albumin and glucose were quantified. Cerebrospinal fluid total protein content and lactate were quantified. Titles of antibodies against echo 9 and Coxsackie A9 and differential cell count were performed. RESULTS: A mean of 555 cells/10 6 L mainly lymphocytes were obtained. Glucose in cerebrospinal fluid was over 50%, serum glucose and lactate levels below 2.1 mmol/L. In the reibergram an absence of intrathecal synthesis was predominant (15/23), IgM synthesis (6/23) and IgM+IgA (2/23). Blood cerebrospinal fluid dysfunction was observed in 15 patients. The mean antibody index was 1,8 for echo 9 and 0,9 for Coxsackie A9. CONCLUSIONS: The intrathecal synthesis pattern of immunoglobulins was different from other enterovirus and from echovirus 9 in non epidemic situations before this epidemic, probably with alteration of viral genome.

[Neuroimmune epidemiological value of reibergram in the first echovirus 16 meningoencephalitis outbreak in Cuba]. / Valor neuroinmunoepidemiologico del reibergrama en la primera epidemia de meningoencefalitis por echovirus 16 en Cuba.

INTRODUCTION: During the third quarter of 2000, an outbreak of echovirus 16 meningoencephalitis was firstly occurred in Cuba and produced vomiting, headache, fever and exanthem that differentiate it from other enterovirus epidemies. OBJECTIVE: To describe the intrathecal immunoglobulin synthesis pattern of the epidemie from the reibergram. PATIENTS AND METHODS: Diagnostic serum and cerebrospinal fluid from 18 children during the acute phase by nephelometrie assay, besides cytochemical and virological study. RESULTS: A predominant absence of intrathecal immunoglobulin synthesis and two patients with IgA and IgM synthesis was produced. 66% of cerebrospinal blood barrier dysfunction, 6.26 10 3 mean albumin ratio, lactate below 2.1 mmol/L and glucose concentration in cerebrospinal fluid was 50% over glucose blood content and 168 cells 10 6 L mainly lymphocytes were observed. CONCLUSIONS: The intratecal immunoglobulin synthesis pattern differs from other enterovirus outbreaks that have affected this population and it seems the one found for the diagnostic period in adults. This finding alerts the possibility of genetic changes in echovirus 16 strain, interesting from the neuroimmunoepidemiological point of view.