Clinical Features of Acute and Fulminant Myocarditis in Children　- 2nd Nationwide Survey by Japanese Society of Pediatric Cardiology and Cardiac Surgery.

BACKGROUND: The 1st nationwide survey by the Japanese Society of Pediatric Cardiology and Cardiac Surgery of acute or fulminant myocarditis (AMC/FMC) in children revealed that the survival rate of FMC was only 51.6%. The 2nd nationwide survey was performed to evaluate the recent outcomes of pediatric myocarditis.Methods and Results:Questionnaires regarding patients aged ≤18 years with AMC/FMC during the period from January 2006 to December 2011 were mailed. A total of 221 cases (age 6.5±5.3 years, 116 boys and 105 girls) were reported. There were 145 (65.6%) and 74 cases (33.5%) of AMC/FMC, respectively; the type of myocarditis was not reported in the remaining 2 cases (0.9%). Viruses were identified in 56 cases (25.3%), including coxsackie B in 9 and influenza A in 8. Histopathology by either endomyocardial biopsy or autopsy was obtained in 38 cases (19.2%). Intravenous immunoglobulin was effective in 49 (34.3%) of 143 cases. Steroid therapy was effective in 20 (32.8%) of 61 cases. Mechanical circulatory support was given in 54 cases (24.4%) and 94.2% of them were patients with FMC. The survival rates for the whole study population, acute myocarditis, and FMC were 75.6%, 91.0%, and 48.6%, respectively. CONCLUSIONS: The survival rate of children with myocarditis was almost identical to that of 10 years ago. (Circ J 2016; 80: 2362-2368).

Colchicine aggravates coxsackievirus B3 infection in mice.

BACKGROUND: There is a clinical need for immunosuppressive therapy that can treat myocarditis patients in the presence of an active viral infection. In this study we therefore investigated the effects of colchicine, an immunosuppressive drug which has been used successfully as treatment for pericarditis patients, in a mouse model of coxsackievirus B3(CVB3)-induced myocarditis. METHODS: Four groups of C3H mice were included: control mice (n=8), mice infected with CVB3 (1×10(5) PFU, n=10), mice with colchicine administration (2mg/kg i.p, n=5) and mice with combined CVB3 infection and colchicine administration (n=10). After three days, the heart, pancreas and spleen were harvested and evaluated using (immuno)histochemical analysis and CVB3 qPCR. RESULTS: Mice were terminated at day 3 post-virus infection as colchicine treatment rapidly resulted in severe illness and mortality in CVB3-infected mice. Colchicine significantly decreased the number of macrophages in the heart in CVB3-infected mice (p<0.01) but significantly increased the number of neutrophils (p<0.01). In the pancreas, colchicine caused complete destruction of the acini in the CVB3-infected mice and also significantly decreased macrophage (p<0.01) and increased neutrophil numbers (p<0.01). In the spleen, colchicine treatment of CVB3-infected mice induced massive apoptosis in the white pulp and significantly inhibited the virus-induced increase of megakaryocytes in the spleen (p<0.001). Finally, we observed that colchicine significantly increased CVB3 levels in both the pancreas and the heart. CONCLUSIONS: Colchicine treatment in CVB3-induced myocarditis has a detrimental effect as it causes complete destruction of the exocrine pancreas and enhances viral load in both heart and pancreas.

Identification of a nucleotide in 5' untranslated region contributing to virus replication and virulence of Coxsackievirus A16.

Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5' untranslated region (5'UTR), have not been investigated until now. Here, a series of nucleotides present in 5'UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5'UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5'UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5'UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5'UTR of CA16, providing a crucial molecular target for antiviral drug development.

Liver X receptor activation enhances CVB3 viral replication during myocarditis by stimulating lipogenesis.

AIMS: Viral myocarditis (VM) is severe cardiac inflammation that can result in sudden death or congestive heart failure in previously healthy adults, with no effective therapy. Liver X receptor (LXR) agonists have both anti-inflammatory and lipid-lowering properties. This study investigates whether LXR agonist T0901317 may modulate viral replication and cardiac inflammation during VM. METHODS AND RESULTS: (i) Adult mice were administered T0901317 or vehicle with the onset of inflammation during CVB3 virus myocarditis or (ii) treated 2 days prior to CVB3 infection. Against what we expected, T0901317 treatment did not alter leucocyte infiltration after CVB3 infection; yet pre-administration with T0901317 resulted in increased mortality upon CVB3 infection, higher cardiac viral presence, and increased cardiomyocyte damage when compared with the vehicle. Furthermore, we show a correlation of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) with CVB3 viral load in the heart and that T0901317 is able to enhance the cardiac expression of FAS and SREBP-1c. Finally, we show in vitro that T0901317 is able to exaggerate CVB3-mediated damage of Vero cells, whereas inhibitors of FAS and the SREBP-1c reduce the viral presence of CVB3 in neonatal cardiomyocytes. CONCLUSION: LXR agonism does not modulate cardiac inflammation, but exacerbates virus-mediated myocardial damage during VM by stimulating lipid biosynthesis and enhancing CVB3 replication.

[A case of congenital Coxsackie B virus infection].

The paper describes a clinical case of congenital Coxsackie B virus infection in a girl aged 1 month and 15 days of life who has undergone a detailed postmortem examination.

[B1a and b1b evolutionary branch of coxsackie virus A16 co-prevailed in Inner Mongolia Autonomous Region].

To study on the molecular evolution of Coxsackie virus A16 (CVA16)isolated from clinical speci-mens of Hand, foot and mouth Disease( HFMD) patients in Inner Mongolia in 2010. A total of 921 clinical specimens including stools, throat swabs and vesicle fluids were collected from 888 HFMD patients in out-patient service in Inner Mongolia and viral isolation was then performed, the positive viral isolates were identified by using the real-time PCR method detecting CVA16. A total of 50 CVA16 isolates were selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly, and the VP1 coding regions of representative CVA16 isolates were amplified and sequenced. Finally the phylogenetic tree was constructed among the VP1 coding regions of the different genotypes and subgenotypes of CVA16 strains. Eighty two viruses were isolated form 921 clinical specimens, the positive rate was 8. 90%, of which 3 viruses were isolated from severe cases and 1 viruses was from death cases. The nucleotide acid of 50 representative CVA16 strains in Inner Mongolia were closed to CVA16 strains isolated from mainland China since 1998, especially from Beijing in 2009 and from Henan in 2010, the identity were 96. 18% approximately 98. 88% and 94. 94a approximately 98. 76%, respectively. There was a little difference in the nucleotide acid between the CVA16 strains from Inner Mongolia in 2010 and in 2007, the identity were 91. 68% approximately 96. 52% The phylogenetic tree showed that all CVA16 strains clustered within Bla and B1b evolution branch of B1 genotype. There was slight difference in the nucleotide and the amino acid in VP1 region among the 50 Inner Mongolia CVA16 strains, the identity were 89. 99% approximately 100% and 98. 31% approximately 100%, respectively, indicating that these strains belonged to many different viral transmission chains. The CVA16 strains circulated in Inner Mongolia in 2010 were all belong to B1a and B1b evolution branch of B1 genotype, and the two evolutionary branchs of Coxsackie virus A16 were co-evolved and co-prevailed in Inner Mongolia Autonomous Region.

[Molecular monitoring of non-polio enteroviruses in European territory of Russia in 2008 - 2011].

As a result of 4 year monitoring the landscape of enteroviruses circulating in European territory of Russia was established to be presented by at least 50 serologic types. Phylogenetic analysis of ECHO30, ECHO9, Coxsackie A9, ECHO6 virus strains that had caused a seasonal increase of aseptic meningitis morbidity in 2008 - 2011 was carried out.

Antiviral activity of coxsackievirus B3 3C protease inhibitor in experimental murine myocarditis.

BACKGROUND: We investigated the efficacy of a 3C protease inhibitor (3CPI) in a murine coxsackievirus B3 (CVB3) myocarditis model. CVB3 is a primary cause of viral myocarditis. The CVB3 genome encodes a single polyprotein that undergoes a series of proteolytic events to produce several viral proteins. Most of this proteolysis is catalyzed by the 3C protease (3CP). METHODS AND RESULTS: By way of a micro-osmotic pump, each mouse received 50 mM 3CPI in 100 µL of 100% dimethyl sulfoxide (DMSO) during a 72-hour period. On the day of pump implantation, mice (n = 40) were infected intraperitoneally with 10(6) plaque-forming units of CVB3. For the infected controls (n = 50), the pump was filled with 100% DMSO without 3CPI. The 3-week survival rate of 3CPI-treated mice was significantly higher than that of controls (90% vs 22%; P < .01). Myocardial inflammation, viral titers, and viral RNA levels were also reduced significantly in the 3CPI-treated group compared with these measures in the controls. CONCLUSIONS: The protein-based drug 3CPI inhibited the activity of 3CP of CVB3, significantly inhibited viral proliferation, and attenuated myocardial inflammations, subsequent fibrosis, and CVB3-induced mortality in vivo. Thus, this CVB3 3CPI has the potential to be a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.

TRIF is a critical survival factor in viral cardiomyopathy.

TRIF is a member of the innate immune system known to be involved in viral recognition and type I IFN activation. Because IFNs are thought to play an important role in viral myocarditis, we investigated the role of TRIF in induced myocarditis in mice. Whereas C57BL/6 (wild-type) mice showed only mild myocarditis, including normal survival postinfection with coxsackievirus group B serotype 3 (CVB3), infection of TRIF(-/-) mice led to the induction of cardiac remodeling, severe heart failure, and 100% mortality (p < 0.0001). These mice showed markedly reduced virus control in cardiac tissues and cardiomyocytes. This was accompained with dynamic cardiac cytokine activation in the heart, including a suppression of the antiviral cytokine IFN-ß in the early viremic phase. TRIF(-/-) myocytes displayed a TLR4-dependent suppression of IFN-ß, and pharmacological treatment of CVB3-infected TRIF(-/-) mice with murine IFN-ß led to improved virus control and reduced cardiac inflammation. Additionally, this treatment within the viremic phase of myocarditis showed a significant long-term outcome indexed by reduced mortality (20 versus 100%; p < 0.001). TRIF is essential toward a cardioprotection against CVB3 infection.

α-Galactosylceramide protects mice from lethal Coxsackievirus B3 infection and subsequent myocarditis.

Myocarditis is an inflammation of the myocardium which often follows virus infections. Coxsackievirus B3 (CVB3), as a marker of the enterovirus group, is one of the most important infectious agents of virus-induced myocarditis. Using a CVB3-induced myocarditis model, we show that injection α-galactosylceramide (α-GalCer), a ligand for invariant natural killer (NK) T (iNK T) cells, can protect the mice from viral myocarditis. After the systemic administration of α-GalCer in CVB3 infected mice, viral transcription and titres in mouse heart, sera and spleen were reduced, and the damage to the heart was ameliorated. This is accompanied by a better disease course with an improved weight loss profile. Compared with untreated mice, α-GalCer-treated mice showed high levels of interferon (IFN)-γ and interleukin (IL)-4, and reduced proinflammatory cytokines and chemokines in their cardiac tissue. Anti-viral immune response was up-regulated by α-GalCer. Three days after CVB3 infection, α-GalCer-administered mice had larger spleens. Besides NK T cells, more macrophages and CD8(+) T cells were found in these spleens. Upon stimulation with phorbol myristate acetate plus ionomycin, splenocytes from α-GalCer-treated mice produced significantly more cytokines [including IFN-γ, tumour necrosis factor-α, IL-4 and IL-10] than those from untreated mice. These data suggest that administration of α-GalCer during acute CVB3 infection is able to protect the mice from lethal myocarditis by local changes in inflammatory cytokine patterns and enhancement of anti-viral immune response at the early stage. α-GalCer is a potential candidate for viral myocarditis treatment. Our work supports the use of anti-viral treatment early to reduce the incidence of virus-mediated heart damage.

Avoiding drug-resistance development by novel approach of combining anti-enteroviral substances against coxsackievirus B1 infection in mice.

Current study presents a novel scheme for combined application of anti-enteroviral substances in coxsackievirus B1 neuroinfection in newborn mice. It consists of a consecutive alternating, not simultaneous, administration of the substances in combination. A triple combination showing good efficacy was selected as a result of a screening of double, triple and quadruple combinations of enteroviral inhibitors. Its effectiveness is expressed in lengthening of the mean survival time and about 50% reduction of mortality rate in infected newborns as compared both to the placebo group, individual compounds used alone every day, and to the same combination applied simultaneously every day. Chronology of alternation of the individual drug administration plays a key role in the efficacy of the combination. Studies of the drug sensitivity of viral brain isolates from mice, treated with the drug combination indicate that virus isolates from the group treated with the alternating combination not only preserve, but even increase their sensitivity to the drugs. MIC(50) values of virus isolates from groups treated with monotherapies of the compounds manifested development of drug resistance. Obviously, the consecutive alternating administration of anti-enteroviral substances hinders the occurrence of drug resistance in the course of experimental coxsackievirus B1 infection in mice.

[Acute myocarditis due to Coxsackie virus B3 in two infants]. / Acute myocarditis door coxsackie B3-virus bij twee zuigelingen.

In the spring and summer of 2008 two seriously ill male infants were admitted to a paediatric intensive care unit. Initially, both had a fever, were drinking less and were pale complexioned. Physical examination revealed tachycardia, slow capillary filling and liver enlargement. Within a few hours, both infants developed circulatory and respiratory failure. A chest radiograph showed that the heart was enlarged and echocardiography revealed that the pump function of both ventricles was severely diminished. Myocarditis caused by Coxsackie virus B3 was diagnosed when the virus was demonstrated in serum and faeces. At the last follow-up, one infant still had severe pump function disorders, and the other one died. Coxsackie virus B3 is a non-polio enterovirus that usually causes mild clinical syndromes but is also associated with myocarditis and overwhelming, systemic neonatal infections. In neonates with mild symptoms one should be alert to progression to circulatory insufficiency, especially if the mother experiences a flu-like illness in the perinatal period. Early recognition of heart failure and adequate diagnostic testing for cardiotropic viruses is important as morbidity and mortality is considerable.

Increased activity of Coxsackievirus B1 strains associated with severe disease among young infants in the United States, 2007-2008.

BACKGROUND: Enterovirus infections are very common and typically cause mild illness, although neonates are at higher risk for severe illness. In 2007, the Centers for Disease Control and Prevention (CDC) received multiple reports of severe neonatal illness and death associated with coxsackievirus B1 (CVB1), a less common enterovirus serotype not previously associated with death in surveillance reports to the CDC. METHODS: This report includes clinical, epidemiologic, and virologic data from cases of severe neonatal illness associated with CVB1 reported during the period from 2007 through 2008 to the National Enterovirus Surveillance System (NESS), a voluntary, passive surveillance system. Also included are data on additional cases reported to the CDC outside of the NESS. Virus isolates or original specimens obtained from patients from 25 states were referred to the CDC picornavirus laboratory for molecular typing or characterization. RESULTS: During 2007-2008, the NESS received 1079 reports of enterovirus infection. CVB1 accounted for 176 (23%) of 775 reported cases with known serotype, making it the most commonly reported serotype for the first time ever in the NESS. Six neonatal deaths due to CVB1 infection were also reported to the CDC during that time. Phylogenetic analysis of the 2007 and 2008 CVB1 strains indicated that the increase in cases resulted from widespread circulation of a single genetic lineage that had been present in the United States since at least 2001. CONCLUSIONS: Healthcare providers and public health departments should be vigilant to the possibility of continuing CVB1-associated neonatal illness, and testing and continued reporting of enterovirus infections should be encouraged.

Outbreak of life-threatening coxsackievirus B1 myocarditis in neonates.

In the summer and fall of 2007, we observed a unique cluster of cases of severe coxsackievirus B1 (CVB1) infection among Chicago area neonates. Eight neonates had closely related strains of CVB1 that were typed at the Centers of Disease Control and Prevention; 2 other neonates had CVB infections, 1 of which was further identified as serotype CVB1. All had severe myocarditis; 1 neonate underwent heart transplantation, and 1 died of severe left ventricular dysfunction.

Hand, foot and mouth disease complicated with central nervous system involvement in Taiwan in 1980-1981.

Sixteen cases from the 1980-1981 Taiwan outbreak of hand, foot and mouth disease (HFMD) associated with central nervous system involvement were identified: nine had polio-like syndrome, four had encephalitis or encephalomyelitis, one had cerebellitis, and two had aseptic meningitis. They all had fever, five (31%) had documented myoclonic jerk, and 15 (93%) had HFMD. Their mean blood leukocyte count was 12,490/microL, and five (31%) had leukocytosis (> 15,000/microL); mean cerebrospinal fluid (CSF) leukocyte count was 156/microL, CSF protein was 57 mg/dL and CSF glucose was 57 mg/dL. Two patients with HFMD plus encephalitis died within 1 day of hospitalization, and one of them had acute cardiopulmonary failure mimicking myocarditis. Twenty years later, at least one male patient had sequelae of polio-like syndrome and was therefore exempted from military service. Clinical severity was comparable to the 1998 EV71 epidemic.

Astragaloside IV exerts antiviral effects against coxsackievirus B3 by upregulating interferon-gamma.

Coxsackievirus B3 (CVB3) is a major pathogen for viral myocarditis and dilated cardiomyopathy in children and young adults. The aim of this study was to determine the antiviral effects of astragaloside IV against CVB3, and the underlying mechanism. First, we evaluated antiviral effects of astragaloside IV in vitro by measuring the virus titers of CVB3 in primarily cultured myocardial cells infected with CVB3, and in vivo by assessing the morbidity, mortality, heart-to-body weight ratio (HW/BW), and virus titers in BALB/c mice infected with CVB3. Then, we performed serum pharmacological experiments by testing the effect of sera from SD rats treated with astragaloside IV on proliferation of CVB3 in primarily cultured myocardial cells. Finally, we determined the effect of astragaloside IV on IFN-gamma mRNA expression in the hearts of infected BALB/c mice. We observed that astragaloside IV decreased virus titers of CVB3 in primarily cultured myocardial cells. Morbidity, mortality, HW/BW, and virus titers all decreased, and necrosis and mononuclear cell infiltration were alleviated in CVB3-infected mice treated with astragaloside IV, compared with those infected mice without the treatment. In addition, proliferation of CVB3 was inhibited by the sera of rats treated with astragaloside IV. Moreover, we observed that IFN-gamma mRNA expression was increased in mice treated with astragaloside IV. Therefore, we conclude that astragaloside IV exerts antiviral effects against CVB3 by upregulating expression of IFN-gamma mRNA.

Quantitative analysis of viral RNA in the murine heart and pancreas with different concentration of coxsackievirus B3.

OBJECTIVES: We investigated the clinical features, pathologic changes, and viral RNA kinetics in the course of acute and subacute experimental coxsackievirus B3 (CVB3) infection in a murine model. METHODS: Five-week-old A/J inbred male mice were divided into 5 groups. Four of those groups were inoculated intraperitoneally with 5 x 10(4) (group 1), 1 x 10(5) (group 2), 5 x 10(5) (group 3), or 1 x 10(6) (group 4) PFU of CVB3. Control mice were inoculated with uninfected Vero cell lysate in DMEM. Mice from each group were sacrificed on days 7 or 14 after inoculation. RESULTS: Bloody diarrhea, earlier weight loss, perianal swelling, and death were correlated with higher viral load. One of ten mice in group 3 and 5 of 10 mice in group 4 died spontaneously between days 4 and 12 after inoculation. All of the remaining 34 mice of infected groups demonstrated extensive pancreatic inflammation. Focal myocarditis developed in only 4 (11.8%) of those 34 subjects. Amylase and creatine kinase activities in the serum were increased in the mice of infected groups. CVB3 RNA was detected in the heart and pancreatic tissue in all subjects. The CVB3 RNA copy number in pancreatic tissue was not correlated with the severity of inflammation. CONCLUSIONS: In the murine model, viral loading dose determines the clinical features of CVB3-induced infection, and the severity of pancreatitis is not correlated with the viral loading dose or tissue level of viral RNA. .

Antiviral effects of sophoridine against coxsackievirus B3 and its pharmacokinetics in rats.

Coxsackievirus B3 (CVB3) is a major pathogen for acute and chronic viral myocarditis. The aim of this study was to investigate the antiviral effects of sophoridine, an alkaloid extracted from Chinese medicinal herb, Sophora flavescens, against CVB3, and the underlying pharmacokinetics. First, we determined the antiviral effects of sophoridine against CVB3 in in vitro (primarily cultured myocardial cells), in vivo (BALB/c mice) and serum pharmacological experiments. Then, we determined the pharmacokinetic behavior in serum samples of SD rats after oral administration by HPLC. Finally, we determined the effects of sophoridine on the production of cytokines in a murine viral myocarditis model by measuring mRNA expression of some important cytokines in hearts of infected BALB/c mice by RT-PCR. We found that sophoridine exhibited obvious antiviral effects both in vitro and in vivo, and serum samples obtained from rats with oral administration of sophoridine reduced the virus titers in infected myocardial cells. The serum concentration profile correlated closely with antiviral activity profile. Moreover, sophoridine significantly enhanced mRNA expression of IL-10 and IFN-gamma, but decreased TNF-alpha mRNA expression. In conclusion, sophoridine possesses antiviral activities against CVB3, by regulating cytokine expression, and it is likely that sophoridine itself, not its metabolites, is mainly responsible for the antiviral activities. Therefore, sophoridine may represent a potential therapeutic agent for viral myocarditis.

Coxsackievirus B3 and the neonatal CNS: the roles of stem cells, developing neurons, and apoptosis in infection, viral dissemination, and disease.

Neonates are particularly susceptible to coxsackievirus infections of the central nervous system (CNS), which can cause meningitis, encephalitis, and long-term neurological deficits. However, viral tropism and mechanism of spread in the CNS have not been examined. Here we investigate coxsackievirus B3 (CVB3) tropism and pathology in the CNS of neonatal mice, using a recombinant virus expressing the enhanced green fluorescent protein (eGFP). Newborn pups were extremely vulnerable to coxsackievirus CNS infection, and this susceptibility decreased dramatically by 7 days of age. Twenty-four hours after intracranial infection of newborn mice, viral genomic RNA and viral protein expression were detected in the choroid plexus, the olfactory bulb, and in cells bordering the cerebral ventricles. Many of the infected cells bore the anatomical characteristics of type B stem cells, which can give rise to neurons and astrocytes, and expressed the intermediate filament protein nestin, a marker for progenitor cells. As the infection progressed, viral protein was identified in the brain parenchyma, first in cells expressing neuron-specific class III beta-tubulin, an early marker of neuronal differentiation, and subsequently in cells expressing NeuN, a marker of mature neurons. At later time points, viral protein expression was restricted to neurons in specific regions of the brain, including the hippocampus, the entorhinal and temporal cortex, and the olfactory bulb. Extensive neuronal death was visible, and appeared to result from virus-induced apoptosis. We propose that the increased susceptibility of the neonatal CNS to CVB infection may be explained by the virus' targeting neonatal stem cells; and that CVB is carried into the brain parenchyma by developing neurons, which continue to migrate and differentiate despite the infection. On full maturation, some or all of the infected neurons undergo apoptosis, and the resulting neuronal loss can explain the longer-term clinical picture.