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Objective: To evaluate the pathogenicity of SSM-CVB3 in a macaque model., Methods: The clinical symptoms of macaques were recorded; hematological, biochemical and histopathological evaluations were completed; viral titers and neutralization titers (NT-titers) in sera were tested; and the mRNA levels of SSM-CVB3, coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF) were determined., Results: After SSM-CVB3 infection, the macaques showed a lack of activity, a poor appetite, a higher body temperature, and severe diarrhea. The macaques also developed hematuria and albuminuria at 4 to 10 days post-inoculation. Virus titers (5.1-6.5 LogTCID(50)/mL) were higher at 6 to 10 days post-inoculation, and NT-titers (6.5-7.3 Log2) reached plateaus at 8 to 14 days post-inoculation. The infected macaques developed serious anemia with decreased RBC and WBC, but the percentages of LYM were increased. The levels of CK, CK-MB, AST and ALT in the sera were 84-169 U/L, 87.6-271.1 U/L, 43-87 U/L and 43-82 U/L, respectively, and all of those were higher than normal. Histological analysis showed obvious cardiac, hepatic and renal damages in the infected macaques and the mRNA contents of SSM-CVB3, CAR and DAF in the heart, liver and kidneys of infected macaques were higher (P<0.05)., Conclusion: This was the first report on experimental SSM-CVB3 infections in macaques with serious hepatic and renal damage, except for myocarditis. The information obtained from this study suggests that the SSM-CVB3 strain and this macaque model could be used for studying CVB3-induced cardiac, hepatic or renal diseases., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: Coxsackievirus B3 (CVB3) is a dominant causative agent for viral myocarditis. So far, effective therapies for the treatment of the disease are not available. 20(S)-Protopanaxtriol is a major component of Panax pseudoginseng and has been clinically used for the treatment of heart diseases. However, it is not known whether 20(S)-protopanaxtriol exerts any anti-viral effects. Thus, the aim of this study was to investigate the therapeutic effects of 20(S)-protopanaxtriol against CVB3 in vivo and in vitro., Methods: The antiviral effects of 20(S)-protopanaxtriol in vitro were evaluated in HeLa cells infected by CVB3. Then, we examined the protective effects of 20(S)-protopanaxtriol on CVB3-induced myocarditis in BALB/c mice. These mice were treated with 20(S)-protopanaxtriol at doses of 100-400 mg·kg(-1)·day(-1) for 7 days and compared with the controls., Results: We found that 20(S)-protopanaxtriol possessed potent antiviral effects on CVB3 in vitro. Compared with control mice, virus titers and pathological changes in the hearts were significantly decreased in the 20(S)-protopanaxtriol-treated group. Furthermore, biochemical markers of myocardial injury such as plasma lactate dehydrogenase and creatine kinase were decreased to normal levels., Conclusions: These data provide the possibility that 20(S)-protopanaxtriol can be used as a potential therapeutic means for treatment of viral myocarditis., (Copyright © 2012 S. Karger AG, Basel.)

Background: Macrophage migration inhibitory factor (MIF) is an upstream regulator in immune and inflammatory responses. However, its role in viral myocarditis remains unknown. In this study, we investigated the role of the MIF in coxsackievirus B3 (CVB3)-induced myocarditis., Methods: Mice were randomized into two groups receiving either Eagle's minimal essential medium (EMEM, control group) or virus solution (infected group). Subsets of mice in the infected group were sacrificed on days 3, 7, 14 and 28 after inoculation. Expression of MIF was detected using an enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction and immunohistochemistry. A neutralizing antibody (Ab) to MIF was injected intraperitoneally from day 0 to 7 after inoculation. Disease severity was estimated by histopathology of the heart and by the heart weight to body weight ratio, and the interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in the myocardium were measured by ELISA on day 14., Results: The serum MIF concentration and expression levels of myocardial MIF mRNA and protein were significantly elevated in mice on days 7 and 14 post-infection. The survival rate was markedly higher and disease severity was obviously less in mice treated with anti-MIF Ab. Furthermore, MIF blockade significantly decreased the IL-1β and TNF-α in the myocarditic heart., Conclusion: These results demonstrate that MIF is an important naturally occurring inflammatory cytokine in CVB3-induced myocarditis, and anti-MIF Ab may lessen the inflammatory response.

Mercury is a widespread environmental contaminant with neurotoxic impacts that have been observed over a range of exposures. In addition, there is increasing evidence that inorganic mercury (iHg) and organic mercury (including methyl mercury) have a range of immunotoxic effects, including immune suppression and induction of autoimmunity. In this study, we investigated the effect of iHg on a model of autoimmune heart disease in mice induced by infection with coxsackievirus B3 (CVB3). We examined the role of timing of iHg exposure on disease; in some experiments, mice were pretreated with iHg (200 μg/kg, every other day for 15 days) before disease induction with virus inoculation, and in others, they were treated with iHg after the acute (viral) phase of disease but before the development of dilated cardiomyopathy (DCM). iHg alone had no effect on heart pathology. Pretreatment with iHg before CVB3 infection significantly increased the severity of chronic myocarditis and DCM compared with control animals receiving vehicle alone. In contrast, treatment with iHg after acute myocarditis did not affect the severity of chronic disease. The increased chronic myocarditis, fibrosis, and DCM induced by iHg pretreatment were not due to increased viral replication in the heart, which was unaltered by iHg treatment. iHg pretreatment induced a macrophage infiltrate and mixed cytokine response in the heart during acute myocarditis, including significantly increased interleukin (IL)-12, IL-17, interferon-γ, and tumor necrosis factor-α levels. IL-17 levels were also significantly increased in the spleen during chronic disease. Thus, we show for the first time that low-dose Hg exposure increases chronic myocarditis and DCM in a murine model.

IL-12 plays an important role in the treatment of many infectious diseases by being administered intravenously or intramuscularly. However, intravenous or intramuscular administration is difficult and inconvenient and may cause side effects. The aim of this study is to develop a novel oral delivery system for IL-12 using genetically engineered Bifidobacterium longum as the carrier and further investigate the efficacy of IL-12-expressed B. longum on the coxsackie virus B3 (CVB3)-induced myocarditis in mice. A mIL-12 gene expression vector pBBADs-IL-12 for B. longum was constructed and transformed into Bifidobacterium. Subsequently, the expression of mIL-12 in the engineered B. longum was identified in vitro by western blot and enzyme-linked immunosorbent assay (ELISA) after l-arabinose induction. Moreover, our data indicated that oral administration of IL-12-expressed B. longum for two weeks after CVB3 infection in the Balb/c mice could downregulate the severity of virus-induced myocarditis, markedly reduce the virus titers in the heart and induce a Th1 pattern in the spleen and heart compared with the controls. In conclusion, a novel oral delivery system of Bifidobacterium for murine IL-12 has been successfully established. Oral administration of mIL-12-transformed B. longum may play a therapeutic role in the treatment of CVB3-induced myocarditis in the mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: To observe the alteration of interleukin-17 and interferon-γ double positive cells (IL-17(+)/IFN-γ(+) cells) in mice with coxsackie virus B3 (CVB3) induced acute viral myocarditis (VMC)., Methods: VMC was induced in male Balb/c mice by peritoneal injection of CVB3. Control mice received PBS injection. At 0, 1, 2, 3, 4 and 6 weeks after injection, pathological scores were determined on hematoxylin-eosin stained heart sections and flow cytometric analysis was performed to evaluate the percent of IL-17(+)/IFN-γ(+) cells among CD4(+) T cells., Results: Compared to control mice, the pathological scores of VMC mice were higher on CVB3 infection week 1 (1.8 ± 0.5), peaked on week 2 (2.8 ± 0.5) and declined thereafter. However, the proportion of IL-17(+)/IFN-γ(+) cells remained steadily at a low level throughout the observation period and was similar between VMC and control mice., Conclusions: Our data shows that IL-17(+)/IFN-γ(+) cells might not be involved in the inflammation process of CVB3 induced VMC mice model.

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.

The most relevant clinical phenotype resulting from chronic enteroviral myocarditis is dilated cardiomyopathy (DCM). Mice of the susceptible mouse strain A.BY/SnJ mimick well human DCM since they develop as a consequence of persistent infection and chronic inflammation a dilation of the heart ventricle several weeks after coxsackievirus B3 (CVB3) infection. Therefore, this model is well suited for the analysis of changes in the heart proteome associated with DCM. Here, we present a proteomic survey of the dilated hearts based on differential fluorescence gel electrophoresis and liquid chromatography-mass spectrometric centered methods in comparison to age-matched non-infected hearts. In total, 101 distinct proteins, which belong to categories immunity and defense, cell structure and associated proteins, energy metabolism and protein metabolism/modification differed in their levels in both groups. Levels of proteins involved in fatty acid metabolism and electron transport chain were found to be significantly reduced in infected mice suggesting a decrease in energy production in CVB3-induced DCM. Furthermore, proteins associated with muscle contraction (MLRV, MLRc2, MYH6, MyBPC3), were present in significantly altered amounts in infected mice. A significant increase in the level of extracellular matrix proteins in the dilated hearts indicates cardiac remodeling due to fibrosis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The most common causes of acute myocarditis and the possible consequence of dilated cardiomyopathy are virus infections. The receptor of the two most common viruses connected to these myocardial diseases was identified as Coxsackie-Adenovirus Receptor. The purpose of this study was to assess Coxsackie-Adenovirus Receptor mRNA expression in the myocardium and search for mutations in the Coxsackie-Adenovirus Receptor gene to compare dilated, inflammatory and ischemic cardiomyopathy with control group. All the myocardial samples were obtained from 35 explanted hearts during heart transplantation, than DNA and RNA were isolated from the muscle samples. cDNA was generated from RNA using reverse transcription, and real-time PCR was performed with relative quantification by β-actin gene as endogenous control. Using DNA extracted from the myocardial samples, we sequenced all the seven exons of the Coxsackie-Adenovirus Receptor gene. Coxsackie-Adenovirus Receptor mRNA expression was higher in both ischemic and dilated cardiomyopathy groups than in inflammatory cardiomyopathy and healthy control groups. Sequencing of CAR gene showed no sign of mutation. Therefore, the sequences result of CAR exons did not show any mutation or polymorphism, that explains a determinant role of CAR in dilated or ischemic CM. Our results suggest that high mRNA expression of Coxsackie-Adenovirus Receptor may support its role in regeneration of the damaged myocardium rather than having any role in viral mediated heart disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate the antiviral effects of the aqueous extract of Spatholobus suberectus Dunn. (A.E.), a Chinese medicinal herb, against coxsackievirus B3 (CVB3)., Methods: The antiviral effects of A.E. against CVB3 in vitro (primarily cultured myocardial cells) and in vivo (BALB/c mice) were determined. Serum pharmacological method was also adopted by in vitro experiments. The effects of A.E. inhibiting the CVB3 mRNA expression were compared by RT-PCR in mice in vivo., Results: A.E. exhibited obvious antiviral: effects in vivo, and serum samples obtained from the rats with oral administration of A.E. (10 μg/mL, 5 μg/mL), reduced the virus titers in the infected myocardial cells (3.00±0.70, 3.55±0.52, P<0.01). Meanwhile, the viral myocarditis induced by CVB3 was inhibited significantly by A.E., and the 15-day mortality was reduced to 40% and 45% (P<0.01) in mice treated with A.E. at doses of 50 mg/kg and 100 mg/kg, respectively, while the 30-day mortality was decreased to 45% and 50%, respectively (P<0.01). Moreover, the mRNA expression of Coxsackie virus B3 was significantly inhibited by A.E., Conclusion: Aqueous extract of Spatholobus suberectus Dunn. (A.E.) has inhibitory effect on CVB3 both in vitro and in vivo.

Infectious disease is frequently cited as a precursor of subsequent autoimmune disease in genetically susceptible hosts. However, the precise mechanisms required for the transition from infection to autoimmunity have not been well defined. We have developed a mouse model of autoimmune myocarditis initiated by infection with Coxsackievirus B3 to trace the cytokine pathways involved. We found that greater production of interleukin-1β (IL-1β) and tumor necrosis factor-α during the early innate response to virus infection is necessary and sufficient to induce a later heart-specific autoimmune disease. Severity of the autoimmune myocarditis is determined by the profile of a number of T helper 1 (Th1) and Th2 cytokines. Th2 responses are especially pronounced in the most severe forms of myocarditis where eosinophils are prominent. The Th1 pathway can lead to infiltration of the heart, but may be dampened by concurrent INF-γ production. Th17 cytokines also contribute to disease, but the signature Th17 cytokine, IL-17A, is not required for cardiac inflammation. Rather, IL-17A is needed for progression to dilated cardiomyopathy. These findings may provide useful markers to identify individuals prone to develop an autoimmune sequel after infection and suggest future early interventions.

Background: The purpose of this study was to establish an ex vivo model of coxsackievirus infection since there seems to be no suitable disease model currently., Methods: Human conjunctival epithelial cells (HCECs) were cultured for 2 weeks in a serum-free air-liquid interface system to produce a multilayered structure. The cells were infected with coxsackievirus A24 (CVA24). Histological changes were investigated by staining the cells with H&E and DAPI, and apoptosis was evaluated using the TUNEL technique. Virus replication was measured in HeLa cells infected with viral progeny from multilayered HCECs, after 1 and 3 days, using the TCID(50) method., Results: Cultured HCECs formed multiple layers. The cells showed characteristics of conjunctival epithelial cells and goblet cells, being immunohistochemically positive for CK19 and MUC5AC, respectively. CVA24 replicated readily in cultured multilayered HCECs. A mild cytopathic effect was noted 1 day after viral inoculation. Cell damage was extensive at 3 days. TUNEL imaging confirmed that the cytopathology was attributable to apoptosis. The TCID(50) data from HeLa cells indicated that the virus was actively replicating at both 1 and 3 days after inoculation., Conclusions: This novel infection model may be useful in investigating the pathogenesis of acute hemorrhagic conjunctivitis and the effectiveness of antiviral treatments.

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.

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

Background: Saffold virus (SAFV) is a newly discovered virus belonging to the genus Cardiovirus of the family Picornaviridae. Using molecular techniques, SAFV has been detected, although infrequently, in the stools of both healthy and diarrheic children and in respiratory specimens collected from children with respiratory disease. The epidemiology and pathogenicity of SAFV remain unclear., Methods: Between July 2009 and October 2010, nasopharyngeal specimens were collected from children with acute respiratory infections. The collected samples were used to isolate respiratory viruses, including coxsackievirus, by cell culture and were tested for SAFV by reverse transcription-polymerase chain reaction., Results: SAFV genotype 2 (SAFV2) was detected in 54 (3.5%) of the 1525 children tested. SAFV2 detections showed an epidemic pattern for a 4-month period with a peak in October 2009. The median age of the SAFV2-positive children was 4 years (range: 7 months-16 years). Among the 35 SAFV2-positive children, excluding cases of viral coinfection, 13 (37.1%) had pharyngitis, 12 (34.3%) had tonsillitis, and 8 (22.8%) had herpangina. Bronchitis and gastroenteritis were detected in 1 case each. Fever (temperature, >38°C) was noted in 33 (94.3%) cases. The median duration of fever was 2 days (range: 1-3 days). Diarrhea was observed in 7 (20.0%) children, but watery and frequent diarrhea was not common. The age distribution and clinical diagnoses associated with SAFV2 infections were similar to those observed with coxsackievirus B4 infections, which detections showed an epidemic pattern during the study period., Conclusion: SAFV2 is a cause of upper respiratory tract illness that exhibits a pathogenicity similar to that of coxsackievirus B.

Background: The IL-23/Th17 pathway is implicated in the pathogenesis of a number of chronic inflammatory and autoimmune diseases. Whether it regulates the viral myocarditis (VMC) is unknown., Results: To examine the pathogenesis role of IL-23/Th17 axis in VMC, we used male BALB/c mice to induced VMC by Coxsackie virus B3 (CVB3) peritoneal injection. IL-23, IL-17, and signal transducer and activator of transcription 3 (STAT3) mRNA in the myocardium of VMC mice were assessed by semi-quantitative RT-PCR. IL-23 and IL-17 protein from blood serum were evaluated by ELISA. Phosphorylated-STAT3 (p-STAT3) protein expression in the myocardium was evaluated by immunohistochemical staining. Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets. Isolated CD4+ T cells from VMC mice were cultured with recombinant IL-23(rIL-23) in vitro. In addition, a STAT3-specific inhibitor (S3I-201) was used to test whether regulation of STAT3 could be partly responsible for Th17 diminution. Results showed that expression of IL-23, IL-17, STAT3 mRNA and protein increased in VMC mice. When purified CD4+ T cells derived from VMC mice were cultured in vitro with rIL-23, the frequency of Th17 cells was dramatically increased, accompanied by significantly enhanced production of IL-17 in the supernatants of cultured CD4+ T cells. S3I-201 significantly restrained Th17 cell proliferation., Conclusions: The IL-23/Th17 pathway axis is strongly expressed in murine VMC, identifying a novel pathway of potential significance in viral myocarditis.

Background: Recently, a new subset of CD4+T helper(Th) cell that predominantly secret cytokine interleukin-9(IL-9) is identified, termed Th9 cell. It has been reported to participate in tissue inflammation and autoimmune responses, and induce disease which differed from Th17 cells. Th17 cells have been shown to play a critical role in viral myocarditis (VMC), but whether Th9 cells are involved in the pathogenesis of VMC remains unclear., Results: BALB/c mice were intraperitoneally (i.p) injected with coxsackie virus B3(CVB3) for establishing VMC models. Control mice were treated with phosphate-buffered saline i.p. On day 0,7,14,21,28,35,42 after injection, myocardial histopathological changes were evaluated by hematoxylin-eosin staining. Splenic Th17 and Th9 cells subsets were analyzed by flow cytometry. And cardiac IL-17, IL-9 mRNA were measured by semi-quantitative reverse transcription-PCR and nested PCR, respectively. Results showed the levels of Th17 cells and IL-17 mRNA obviously increased in VMC mice on 7 day after infection, peaked on day 28, and highly persisted to at least day 42 (p < 0.05). While the frequencies of Th9 cells and IL-9 mRNA showed no significant difference between VMC and control group throughout the course of the experiment(p > 0.05)., Conclusions: It was differentiated Th17 but not Th9 cells significantly elevated in the development of CVB3-induced VMC. The microenvironment of VMC seemed to contribute to the differentiation and proliferation of Th17 rather than Th9 cells. Our preliminary data implied Th9 cells could not protect against VMC nor promote the disease.

Background: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis., Methodology/principal Findings: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression., Conclusions: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.

Both coxsackievirus B3 (CVB3) infection and selenium (Se) deficiency play a pivotal role in Keshan disease of the heart. The Se deficiency was known to contribute to the CVB3-induced myocarditis in acute and subacute phase of infection. However, its effect on the myocarditis in chronic phase of infection has not been examined yet. To address this question, we kept mice on a Se-replete or Se-deficient diet for 28 days, infected them intraperitoneally with CVB3 and maintaining previous diets, we examined them for next 90 days for several parameters indicative of the infection or disease. We found out that the mice on the Se-deficient diet exhibited a higher mortality, lower serum glutathione peroxidase (GPx) activity, evident histopathological changes indicative of myocarditis, and a higher level of viral RNA in the heart. Summing up, these data suggest that the Se-deficiency creates a chronic myocarditis-prone condition by fostering the active virus replication.

The effect of NO-metoprolol, that is, 3-nitrooxypivaloyl metoprolol-amide, a novel NO-releasing derivative of the β1-blocking drug metoprolol was investigated in A.CA/SnJ mice infected with coxsackievirus B3 (CVB3) and compared to metoprolol and placebo. Daily treatment of mice with the respective drug started immediately (experiment A) or 3 days after virus infection (experiment B) and was continued until day 13 post-infection (p.i.). Two doses of NO-metoprolol were administered. Body mass differences, viral load, and histopathological signs of myocarditis were compared between the several groups. As a result, NO-metoprolol diminished significantly the body weight loss, the viral load and the histopathology, whereas metoprolol treatment led solely to a significant attenuation of myocardial damage. In experiment A, low dose NO-metoprolol decreased significantly enteroviral copy numbers. Both doses of NO-metoprolol had a significant effect on reduction of myocardial infiltrates and fibrosis. The data suggest that delayed drug administration might more advantageous. Both doses of NO-metoprolol reduced significantly the scores of four tested parameters compared to placebo. Body weight loss, virus titers, plus-strand as well as minus-strand enteroviral RNA levels, infiltration and fibrosis scores were diminished significantly when NO-metoprolol was given 3 days p.i. In addition, a significant difference regarding the enteroviral copy numbers was observed between low dose NO-metoprolol- and metoprolol-treated mice. Treatment with metoprolol reduced insignificantly the viral load and body weight loss (experiment A and B) but led to a significant reduction of myocardial histopathology in experiment A. The results indicate that NO-metoprolol treatment has a greater therapeutic benefit than metoprolol.

Introduction: CD40 and CD40L, as costimulatory molecules, are reported to play a critical role for cytokine production and antigen-specific T-cell activation in acute viral myocarditis (VMC). The purpose of this study was to probe the therapeutic effect of CD40-Ig in coxsackievirus B3 (CVB3)-induced myocarditis., Methods: Adolescent male Balb/c mice were infected with cardiovirulent CVB3 and then injected with CD40-Ig (0.1 mg/kg) at 6 h and 3 days postinfection (pi), with IgG (0.1 mg/kg) as control. The surviving mice were sacrificed on the seventh day. The copies of CVB3 mRNA in the myocardium were detected by real-time PCR and the expression of CD40 protein and CD4(+) T cell was assessed by immunohistochemistry method. The serum level of IL-4 and IFN-γ and their transcriptions in the myocardium were determined by ELISA and real-time PCR., Results: The mortality was low in VMC model mice receiving CD40-Ig treatment with relived inflammation and reduced transcription of CVB3 in the myocardium. The expression pattern of IFN-γ (Th1-related cytokine) and IL-4 (Th2-related cytokine) was altered to Th1 prevalence in the acute phase of VMC. Intervention of CD40-Ig switched the balance of Th1/Th2., Conclusions: Our findings suggest that intervention of CD40-Ig can relieve the myocardial injury and inhibit viral replication of CVB3 in VMC. Its mechanism may involve blocking the interaction between CD40 and CD40L and regulating the Th1/Th2 balance., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In the present study, we determined the therapeutic efficacy of atorvastatin on Coxsackievirus B3m (CVB3m)-induced myocarditis. Mice were administered Eagle minimal essential medium, virus solution, atorvastatin, or virus plus atorvastatin. Atorvastatin was given 3 days after viral challenge, and the treatment lasted for 14 days. On days 3, 7, 10, 14, 21, and 30 after virus inoculation (same days for the atorvastatin only group), echocardiograms were performed, and blood samples were collected for cardiac troponin I analysis. Myocardial inflammation, cell apoptosis, and Fas expression were detected by histology and immunohistochemistry. Hematoxylin and eosin staining and transmission electron microscopy revealed significant improvement of quantitative pathological features in the CVB3m-infected group treated with atorvastatin. Immunohistochemistry also showed a marked decrease in apoptosis of cardiac cells in the atorvastatin-treated group compared with infected animals without treatment. The differences in the values of cardiac troponin I between the atorvastatin treated and untreated virus-challenged mice were statistically significant (P < 0.05). Reverse transcription-polymerase chain reaction and western blotting revealed that the virus induced marked increases in Fas messenger RNA and protein expression, which was reversed by atorvastatin. These results demonstrate that atorvastatin reduces the histological and functional severity of CVB3m-induced myocarditis and inhibits apoptosis and Fas expression in the myocardium of type B Coxsackie virus-infected mice.

Background: c-fos may play a role in the pathogenesis of some diseases. The expression and function of c-fos in viral myocarditis (VMC) have not yet been reported. To study the change and significance of proto-oncogene c-fos in VMC is the objective of this experiment., Methods: An animal model of VMC was established via coxsackie virus B3 inoculation. VMC mice were then treated with a c-fos monoclonal antibody and isoproterenol and the protein and mRNA expression of c-fos were studied via immunohistochemical analysis and in situ hybridization. Results were simultaneously analyzed for the significance of c-fos expression in mice with VMC., Results: Myocardial necrosis and cell infiltration decreased after treatment with c-fos monoclonal antibody compared to control mice, while myocardial necrosis and cell infiltration were increased after treatment with isoproterenol. Positive cardiomyocytes with c-Fos expression increased at 3, 5, 7, 9, and 15 days after virus inoculation in VMC mice compared to control mice, while returning to almost normal levels at 35 days. The expression level of c-fos mRNA at 3 and 7 days after virus inoculation in VMC mice was also higher than that of control mice., Conclusions: c-fos expression in the cardiomyocytes of VMC mice is significantly increased, c-fos plays an important role in myocardial lesions. The apparent increase in expression of c-fos is likely to be involved in the pathogenesis of VMC.

Coxsackievirus B3 (CVB3) induces myocarditis, an inflammation of the myocardium, in C57Bl/6 male mice but not in mice lacking γδ+ T cells [γδ knockout (γδKO)]. Suppression of myocarditis in γδKO mice corresponds to an increase in CD4(+) CD25(+) FoxP3(+) T regulatory cells. A subpopulation of the T regulatory cells in infected γδKO mice expressed high levels of CD1d, a non-classical major histocompatibility complex class 1-like molecule. Adoptive transfer of CD1d(+) and CD1d(-) CD4(+) CD25(+) cells into infected C57Bl/6 recipients showed that the CD1d(+) subpopulation is substantially more suppressive than the CD1d(-) subpopulation. T cells expressing the γδ T-cell receptor comprised approximately 30-50% of the infiltrating lymphoid cells in the hearts of myocarditic C57Bl/6 mice and approximately half of the γδ+ cells expressed the Vγ4 T-cell receptor. The Vγ4+ cells lysed T regulatory cells from γδKO mice but not from wild-type (C57Bl/6) animals. Lysis was inhibited by antibody to CD1d and zVAD-fmk, a pan-caspase inhibitor. The Vγ4-γδ+ cells were not lytic to T regulatory cells and did not promote myocarditis. These results demonstrate that Vγ4+ cells selectively abrogate T regulatory cells through recognition of CD1d expressed on the regulatory cells and caspase-dependent apoptosis., (© 2010 The Author. Immunology © 2010 Blackwell Publishing Ltd.)

Acute viral myocarditis (AVMC) is characterized by virus-triggered myocardial inflammation, and Coxsackievirus B3 (CVB3) is the primary pathogen. We previously proved that Th17 cells, besides having proinflammatory effects, were involved in AVMC by enhancing humoral response. However, the relationship between Th17 cells and CVB3 replication remains unknown. In this experiment, we infected BALB/c mice with CVB3 for establishing AVMC models and then found that, with the increase of viral replication, the expressions of splenic Th17 cells, serum IL-17, and cardiac IL-17 mRNA were elevated significantly, accompanied by the progressive cardiac injuries of AVMC. Furthermore, on day 5, the peak time for viral replication, correlation was positive between cardiac IL-17 mRNA and CVB3 RNA (correlation index = 0.835; p < 0.01). Although the expressions of Th1 and CD8(+) T cells, which could secrete the antiviral cytokine IFN-γ and damage the heart, were also elevated, along with Th17 cells, in AVMC, the neutralization of IL-17 further upregulated the percentages of splenic Th1 and CD8(+) T cells and the levels of cardiac IFN-γ mRNA. The cardiac pathological changes were obviously improved after neutralization, with reduced viral replication followed by decreases in the cardiac inflammatory cytokines IL-17, TNF-α, and IL-1β. These data suggest that Th17 cells contribute to CVB3 replication in AVMC, and that IL-17 might be an important target for regulating the balance of antiviral immunities.

Coxsackieviruses are important human pathogens, and their interactions with the innate and adaptive immune systems are of particular interest. Many viruses evade some aspects of the innate response, but coxsackieviruses go a step further by actively inducing, and then exploiting, some features of the host cell response. Furthermore, while most viruses encode proteins that hinder the effector functions of adaptive immunity, coxsackieviruses and their cousins demonstrate a unique capacity to almost completely evade the attention of naive CD8(+) T cells. In this artcle, we discuss the above phenomena, describe the current status of research in the field, and present several testable hypotheses regarding possible links between virus infection, innate immune sensing and disease.

Objective: to observe the alteration of T helper cells 17(Th17) in mice with acute viral myocarditis (VMC) induced by coxsackie virus B3 (CVB3), explore the role of Th17 in mice VMC., Methods: CVB3 or PBS was peritoneally injected to Balb/c male mice. Pathological scores were determined in hematoxylin-eosin stained sections and flow cytometric analysis was used to evaluate the frequencies of Th17 subsets in CD4(+) T cells on 7, 14, 21, 28 and 42 days after virus injection., Results: there were significant difference of the pathological scores between the VMC mice and the control ones (P < 0.05). The pathological scores of 7 d VMC subgroup were higher (1.8 ± 0.5) than those of 0 d VMC subgroup, and the scores of 14 d subgroup were highest (2.8 ± 0.4) among the six subgroup of VMC mice, and then showed a decline tendency from 21 d group. Statistical difference of the proportion of Th17 cells were seen between the VMC and controls on different time points (P < 0.05). When compared with the 0 d VMC subgroup the proportion of spleen Th17 cells increased in 7 d VMC subgroup [(2.23 ± 0.89)%], and peaked on 28 d [(5.00 ± 0.81)%]. The results of Th17 proportion were lower than those of the 28 d subgroup., Conclusions: our data show that differentiated Th17 cells might be involved in the inflammation process of CVB3 induced VMC in mice.

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3beta (MAP1LC3beta). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3beta fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.

Enteroviral myocarditis displays highly diverse clinical phenotypes ranging from mild dyspnoea or chest pain to cardiogenic shock and death. Despite detailed studies of the virus life cycle in vitro and in vivo, the molecular interplay between host and virus in disease progression is largely unresolved. Murine models of Coxsackievirus B3 (CVB3)-induced myocarditis well mimic the human disease patterns and can thus be explored to study mechanisms leading from acute to chronic myocarditis. Here, we present a 2-D gel-based proteomic survey of the changes in the murine cardiac proteome that occurs following infection with CVB3. In total, 136 distinct proteins were affected. Proteins, which are involved in immunity and defense and protein metabolism/modification displayed pronounced changes in intensity not only during acute but also at later stages of CVB3 myocarditis. Proteins involved in maintenance of cell structure and associated proteins were particularly influenced in the acute phase of myocarditis, whereas reduction of levels of metabolic enzymes was observed in chronic myocarditis. Studies about changes in protein intensities were complemented by an analysis of protein phosphorylation that revealed infection-associated changes in the phosphorylation of myosin binding protein C, atrial and ventricular isoforms of myosin regulatory light chain 2, desmin, and Rab GDP dissociation inhibitor beta-2.

Background/purpose: Coxsackievirus A2 (Cox A2) was the predominant serotype in the enterovirus outbreak in Taiwan, 2008. However, detailed clinical features of Cox A2 infection have not been reported. In this study, we compared Cox A2 with enterovirus 71 (EV71) in terms of clinical manifestation and epidemiology during the 2008 enterovirus outbreak in Taiwan., Methods: A total of 280 hospitalized patients (97 with culture-proven EV71 infection and 183 with culture-proven Cox A2 infection) in 2008 at the Chang Gung Children's Medical Center were enrolled in this study. Epidemiologic data, clinical manifestations, and outcomes for these patients were collected and compared., Results: Both Cox A2 and EV71 serotypes peaked in June and declined soon afterwards. Seventy-one percent of the patients were younger than 3 years of age. Both groups had the same male-to-female ratio of 1.6:1. Patients with EV71 infection had a significantly longer hospitalization period (4.1 vs. 3.0 days, p< 0.001). Fever, fever for more than 3 days with a temperature above 39 degrees C, lethargy, poor activity, poor appetite and a myoclonic jerk were significantly associated with EV71 infection. Fever, or fever with a temperature above 39 degrees C, febrile seizure, elevated white cell counts, and elevated serum C-reactive protein concentrations were significantly associated with Cox A2 infection. Most patients with EV71 infection presented with hand-foot-mouth disease (78.3%), while most Cox A2-infected patients presented with herpangina (83.6%). Central nervous system complications were found in 18.6% of EV71-infected children, but only in 1.1% of Cox A2-infected children. All the patients with Cox A2 infection showed total recovery. One patient with EV71 infection died from encephalitis with cardiopulmonary failure, and 6.2% of EV71-infected children had neurologic sequelae., Conclusion: Both Cox A2 and EV71 serotypes accounted for the enterovirus outbreak in Taiwanese children in 2008. Compared with those infected by EV71, the children with Cox A2 infection mostly presented with herpangina, had fewer central nervous system complications, and had better overall outcome., (Copyright 2010 Taiwan Society of Microbiology. Published by Elsevier B.V. All rights reserved.)

Aim of Study: The heart-protective effect and mechanism of Qishaowuwei formula (QSW), a Traditional Chinese Medicine formula composed of Radix Astragali, Radix Paeoniae Rubra and Fructus Schisandrae was investigated on murine model of viral myocarditis (VMC) induced by Coxsackievirus B3 (CVB3)., Materials and Methods: Mice were randomly divided into infected control group, QSW high dose group, QSW medium dose group, QSW low dose group and Vitamin C plus Ribavirin treatment group. 50 mice were included in each group. The day of virus inoculation was defined as day 0 and the drug treatment continued once a day for 14 days. Mice were sacrificed on days 3, 7, 14, 21 postinoculation (p.i.). The histopathological changes of myocardium, CVB3 RNA copies in the myocardium, cardiomycytic apoptosis, the serum level of superoxide dismutase (SOD) and maleic dialdehyde (MDA) and the phenotype of T lymphocytes subsets in peripheral blood was analyzed., Results: QSW treatment significantly increase the survival rate (p<0.05) in VMC model. Histopathology and flow cytometry inspection revealed low ratio of cardiomyocytes necrosis and apoptosis in QSW treated mice with dose dependent manner. The cardiomyocytic ultra-structure observed by transmission electron microscope also supported the above results. The ameliorated tissue damage was consistent with reduced CVB3 copy numbers detected by real-time PCR in the myocardium of QSW treated mice. The antioxidant effect of QSW was proved by elevated activity of SOD and reduced level of MDA in the serum. Furthermore, the disturbed balance of CD4+ and CD8+ subsets in peripheral blood was restored., Conclusion: These results demonstrated QSW had potent protective effect against CVB3-induced heart injury and this effect might be mediated by its inhibition on viral replication, antioxidant activity and immunoregulation mechanism., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Coxsackievirus B3 (CVB3) induces cardiac inflammation (myocarditis) in male but not female C57BL/6 mice. Protection of females correlates with reduced expression of TNF-alpha and IL-1beta at both the mRNA and protein levels in the heart. Treatment of females with 300 ng/mouse of recombinant TNF-alpha on days +1 and +3 relative to infection restores myocarditis susceptibility to levels approximating those of infected male mice, showing that TNF-alpha deficiency is central to disease resistance. Female mice express little CD1d on spleen lymphocytes or cardiac myocytes, while females treated with TNF-alpha show increased CD1d expression in both cell populations. TNF-alpha treatment of male or female CD1d knockout (CD1dKO) mice failed to restore myocarditis susceptibility, demonstrating that of the multiple potential TNF-alpha activities, its ability to upregulate this non-classical major histocompatibility complex antigen is its dominant function in myocarditis susceptibility. Bone marrow chimeric mice were produced between female C57BL/6 and C57BL/6 CD1dKO mice so that either hematopoietic or non-hematopoietic cells were CD1d deficient. TNF-alpha treatment of chimeric mice having wild-type (CD1d+) hematopoietic cells restored myocarditis susceptibility, while TNF-alpha treatment of chimeric mice having CD1dKO hematopoietic cells, but CD1d+ myocytes, failed to develop myocarditis, showing that CD1d expression in lymphoid cells controls disease susceptibility.

Aim: To investigate the role of anti-perforin neutralizing antibody in viral myocarditis., Methods: We divided 45 Balb/c mice randomly into 3 groups, a normal control group, a control group inoculated with coxsackie virus B3, and a group inoculated with anti-perforin neutralizing antibody. The second group was inoculated with 0.15 milliliters coxsackie virus B3, and the third group additionally with 0.1 milligrams/kilogram anti-perforin neutralizing antibody at time points of 6 hours and 3 days after infection. Histopathology was performed using haematoxylin and eosin, with apoptosis examined by the terminal transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick, end-labeling method, or Tunel. The expression of caspase-3 in myocardium was investigated by immunohistochemistry and reverse-transcription polymerase chain reaction., Results: The pathologic score, myocardial viral titers, average percentages of apoptotic cardiomyocytes, expression of active caspase-3 protein and messenger ribonucleic acid in the myocardium of the mice receiving anti-PFP neutralizing antibody therapy were all significantly reduced when compared to values from the group inoculated with coxsackie virus B3. The rates of expression of Caspase-3 and myocardial apoptosis were positively correlated with the scores for myocardial pathology., Conclusion: Our results suggest that anti- perforin neutralizing antibody can reduce the myocardial damage by blocking the perforin/granzyme pathway, and downregulating the expression of messenger ribonucleic acid and protein of Caspase-3. These approaches may offer promising novel therapeutic strategies for the clinical treatment of viral myocarditis.

NK4 may be a promising agent to inhibit tumor invasion and metastasis. To observe the effects of NK4 on the cardiovascular system with pathological injury and to discuss the mechanism, we established an experimental model of viral myocarditis (VCM) by coxsackievirus B3 infection in Balb/c mice on Day 0 and administered NK4 twice daily to the VCM and control mice from Day 20 to Day 45. We then evaluated the cardiac function by means of ultrasonic inspection. Hepatocyte growth factor, TNF (tumor necrosis factor)-alpha, and angiotensin II levels in the myocardial tissue were measured with enzyme-linked immunosorbent assay. Myocardium histopathology was examined with hematoxylin and eosin stain. Collagen deposition of the myocardium was detected through Masson staining. Microvessel staining with the RECA antibody and apoptosis detection with terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling were performed in the myocardium. The changes in MMP3 (matrix metalloproteinase 3), MMP9, TIMP1 (tissue inhibitor of metalloproteinase 1), and TGF (transforming growth factor)-beta1 expression in the myocardium were measured by reverse-transcriptase polymerase chain reaction. We found that NK4 intervention increased TGF-beta and angiotensin II expression, suppressed MMPs, improved the activities of TIMPs, and then promoted collagen deposition in the myocardium. NK4 intervention also decreased the microvessels' density and increased the apoptotic cell count in the myocardia of VCM mice. However, we did not observe the obvious changes in the myocardia of control mice after NK4 intervention. These data suggest that NK4 made negative impacts on the restoration of cardiac function and the recovery from VCM in the experimental mice.

Background: Acute heart failure is a potentially fatal manifestation of viral myocarditis. Development of myocardial damage in myocarditis involves cardiomyocyte apoptosis. Levosimendan is a novel calcium sensitizing inotropic agent with anti-apoptotic properties. We studied the feasibility of inotropic treatment with levosimendan and its effects on apoptosis in experimental acute heart failure caused by coxsackievirus myocarditis., Materials and Methods: Adolescent BALB/c mice were infected with myocarditic Woodruff variant of coxsackievirus B3 (2 x 10(4) plaque-forming units). Mice were randomized into those receiving levosimendan 0.33 mg kg(-1) (total dose 1 mg kg(-1) day(-1)) (n = 20) or vehicle (n = 19) given orally by gauge three times a day for 7 days after infection. Left ventricular function was evaluated by transthoracic echocardiography and the mice were euthanized on day 7. Histopathology, amount of virus in the heart (virus titration assay) and cardiomyocyte apoptosis (TUNEL assay) were studied. Uninfected untreated control mice were also studied., Results: Infection resulted in histopathologically severe myocarditis and significant impairment of left ventricular function. Levosimendan treatment significantly improved ventricular function (fractional shortening 0.32 +/- 0.04 vs. 0.23 +/- 0.05, P = 0.005; contractility 0.60 +/- 0.12 vs. 0.39 +/- 0.14, P = 0.007 and myocardial performance index 0.36 +/- 0.06 vs. 0.62 +/- 0.15, P < 0.0001) compared with vehicle. Levosimendan also reduced cardiomyocyte apoptosis (0.26 +/- 0.08% vs. 0.44 +/- 0.15% in vehicle, P = 0.008), but did not have an effect on areas of myocardial necrosis or inflammation, or the amount of virus in the heart. Levosimendan treatment did not affect mortality (total mortality 63%). CONCLUSIONS; Levosimendan improves ventricular function and inhibits cardiomyocyte apoptosis; therefore, it is suggested as a potentially feasible therapy in acute heart failure caused by viral myocarditis.

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.

During fall 2008, an outbreak of hand, foot, and mouth disease (HFMD) with onychomadesis (nail shedding) as a common feature occurred in Finland. We identified an unusual enterovirus type, coxsackievirus A6 (CVA6), as the causative agent. CVA6 infections may be emerging as a new and major cause of epidemic HFMD.

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.

Coxsackieviruses are significant human pathogens, and the neonatal central nervous system (CNS) is a major target for infection. Despite the extreme susceptibility of newborn infants to coxsackievirus infection and viral tropism for the CNS, few studies have been aimed at determining the long-term consequences of infection on the developing CNS. We previously described a neonatal mouse model of coxsackievirus B3 (CVB3) infection and determined that proliferating stem cells in the CNS were preferentially targeted. Here, we describe later stages of infection, the ensuing inflammatory response, and subsequent lesions which remain in the adult CNS of surviving animals. High levels of type I interferons and chemokines (in particular MCP-5, IP10, and RANTES) were upregulated following infection and remained at high levels up to day 10 postinfection (p.i). Chronic inflammation and lesions were observed in the hippocampus and cortex of surviving mice for up to 9 months p.i. CVB3 RNA was detected in the CNS up to 3 months p.i at high abundance ( approximately 10(6) genomes/mouse brain), and viral genomic material remained detectable in culture after two rounds of in vitro passage. These data suggest that CVB3 may persist in the CNS as a low-level, noncytolytic infection, causing ongoing inflammatory lesions. Thus, the effects of a relatively common infection during the neonatal period may be long lasting, and the prognosis for newborn infants recovering from acute infection should be reexplored.

Rationale: Myocardial infiltrating macrophages play an important role in the pathogenesis of viral myocarditis in male BALB/c mice following coxsackievirus B3 (CVB3) infection. Interestingly, comparable macrophage numbers were observed in the myocardium of female mice during acute myocarditis., Objective: Given CVB3 infection causes severe myocarditis in male but not female mice, we postulated that macrophages infiltrating the myocardium of female mice may display distinct functional properties that contribute to differential susceptibility to CVB3 myocarditis., Methods and Results: Here, we found that myocardial infiltrating macrophages from CVB3-infected male mice expressed high levels of classically activated macrophages (M1) markers, including inducible nitric oxide synthase, interleukin-12, tumor necrosis factor-alpha, and CD16/32, whereas those of females showed enhanced expression of arginase 1, interleukin-10, macrophage mannose receptor (MMR) and macrophage galactose type C-type lectin (MGL) that were associated with alternatively activated macrophage (M2) phenotype. Moreover, distinct myocardial-derived cytokines were found to play a critical role in differential macrophage polarization between sexes after CVB3 infection. Adoptive transfer of ex vivo programmed M1 macrophages, as expectedly, significantly increased myocarditis in both male and female mice. Strikingly, transfer of M2 macrophages into susceptible male mice remarkably alleviated myocardial inflammation by modulating local cytokine profile and promoting peripheral regulatory T cell (Treg) differentiation., Conclusions: Taken together, this study may facilitate the understanding of the mechanism underlying gender bias in susceptibility to CVB3 myocarditis and the development of therapeutic strategies based on macrophage polarization for inflammatory heart disease.

Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice which depends upon CD4(+) T helper type 1 [Th1; i.e. interferon-gamma(+) (IFN-gamma(+))] and gammadelta(+) cells. Depleting gammadelta(+) cells using anti-gammadelta antibody suppresses myocarditis and CD4(+) IFN-gamma(+) cell numbers in the spleen and heart of infected mice while increasing CD4(+) FoxP3(+) cells. Mice deficient in gammadelta(+) cells have increased numbers of naïve (CD44(lo) CD62L(hi)) and fewer effector (CD44(hi) CD62(lo)) memory CD4(+) cells than infected gammadelta(+)-cell-sufficient mice. Virus neutralizing antibody titres are not significantly different between gammadelta(+) T-cell-sufficient and -deficient animals. To confirm that the memory cell response differs in acutely infected mice lacking gammadelta(+) cells, CD4(+) cells were purified and adoptively transferred into naïve recipients, which were rested for 4 weeks then infected with CVB3. Recipients given either 0.5 x 10(6) or 1.0 x 10(6) CD4(+) from infected donors developed over twice the severity myocarditis and 10-fold less cardiac virus titre compared with recipients given equivalent numbers of CD4(+) cells from infected and gammadelta(+)-cell-depleted donor animals. Additionally, to show that more functionally active T regulatory cells are present in gammadelta(+) T-cell-depleted mice, CD4(+) CD25(+) and CD4(+) CD25(-) cells were isolated and adoptively transferred into infected recipients. Mice receiving CD4(+) CD25(+) cells from gammadelta(+) T-cell-depleted donors developed significantly less myocarditis and CD4(+) Th1 cell responses compared with mice receiving equal numbers of CD4(+) CD25(+) cells from infected gammadelta(+) T-cell-sufficient animals. This study shows that gammadelta(+) cells promote CD4(+) IFN-gamma(+) acute and memory responses by limiting FoxP3(+) T regulatory cell activation.

Background: A 5-year-old female Sichuan snub-nosed monkey died at the zoological garden from infection with coxsackievirus B3., Methods: The diagnosis was made on the basis of pathologic features, immunohistochemistry, microbiological detection, and reverse transcription-polymerase chain reaction (RT-PCR)., Results: Histologic examination of formalin-fixed, paraffin-embedded tissues revealed a severe degree of predominantly lymphocytic infiltration of the cardiac muscle. Picornaviridae-like virions were found in the supernatants of cardiac muscle tissues homogenates, in the pericardial fluid, and in Vero cell cultures, by electron microscopy. Coxsackievirus B3 particles were detected in cardiac muscle cells by immunofluorescence. RT-PCR performed on an extract of cardiac muscle tissue revealed a DNA sequence specific for coxsackievirus B3., Conclusions: This is the first report of a Sichuan snub-nosed monkey dying from a virus.

Background: Autoantibodies against various endogenous proteins are found in myocarditis. Troponin autoantibodies are detected in patients with chronic dilated cardiomyopathy, but their presence in myocarditis remains unknown. We set out to study the presence of troponin autoantibodies in experimental viral myocarditis., Materials and Methods: BALB/c mice infected with coxsackievirus B3 Nancy strain were followed-up at days 1-7 and 2, 4, 8 and 12 weeks after infection. Levels of circulating cardiac troponin I and circulating troponin autoantibodies were measured. Transthoracic echocardiography was performed. Myocarditis was histopathologically graded and cardiomyocyte apoptosis was quantified (TUNEL)., Results: Histopathologically relatively mild acute myocarditis followed by persistent cardiomyocyte damage was observed. Rate of cardiomyocyte apoptosis was the highest on day 5 (0.16 +/- 0.01% vs. 0.03 +/- 0.01% in controls, P < 0.001). Circulating troponin I levels were increased to day 5 (45.2 +/- 6.5 ng mL(-1), P < 0.005 vs. controls). Troponin autoantibodies were detected from 2 weeks after infection (20% of animals had autoantibodies at 2 weeks, 60% at 4 and 8 weeks and 20% at 12 weeks, P < 0.05 vs. controls). Fractional shortening remained decreased after acute myocarditis (0.36 +/- 0.02 at 4 weeks, 0.30 +/- 0.02 at 8 and 12 weeks vs. 0.41 +/- 0.01 before infection, P < 0.01) parallel to development of troponin autoantibodies., Conclusion: Troponin autoantibodies are formed in experimental virus induced myocarditis following troponin I release and cardiomyocyte apoptosis. The definite role of these autoantibodies remains to be further characterized.

The characteristics of dilated cardiomyopathy (DCM) resulting from chronic viral myocarditis are remodeling processes of the extracellular matrix. Based on our findings of enhanced osteopontin (OPN) expression in inflamed human hearts, we further investigated in the murine model of acute and chronic coxsackievirus (CV)B3-myocarditis the role of OPN regarding its involvement in resolution of cardiac virus infection and fibrosis. In hearts of A.BY/SnJ mice susceptible to chronic CVB3-myocarditis, a pronounced increase of OPN expression levels was detected by microarray analysis and quantitative RT-PCR during acute stages of myocarditis. Combined immunohistochemistry and in situ hybridization identified infiltrating macrophages as main OPN producers. In contrast to resistant C57BL/6 and OPN gene-deficient mice, transcription levels of matrix metalloproteinase-3, TIMP1 (tissue inhibitor of metalloproteinases-1), uPA (urokinase-type plasminogen activator), and transforming growth factor beta1 were elevated in susceptible mice, and as a consequence, procollagen-1alpha mRNA expression and fibrosis was considerably enhanced. Treatment of infected susceptible mice with the vitamin D analog ZK 191784 led to decreased myocardial expression levels of OPN, metalloproteinase-3, TIMP1, uPA, and procollagen-1alpha and subsequently to reduced fibrosis. Concurrently, the fibrosis-relevant signaling molecules pERK (phosphorylated extracellular signal-regulated kinase) and pAkt (phosphorylated Akt), increased in A.BY/SnJ mice, were diminished in ZK 191784-treated mice. Here, we show that high expression levels of OPN in acute myocarditis are associated with consecutive development of extensive fibrosis that can be reduced by treatment with a vitamin D analog. Thus, OPN may serve as a diagnostic tool as well as a potential therapeutic target to limit cardiac remodeling in chronic myocarditis.

Infection with coxsackievirus B4 (CVB4) may result in an acute severe necrotizing pancreatitis that mostly remains restricted to the acini of the exocrine parenchyma. The mechanisms responsible for this tissue damage, however, remain poorly understood. We here report that COAM, a polyanionic carboxylic acid, provides marked protection against CVB4-induced pancreatitis in a mouse model. Despite the fact that COAM largely reduced disease severity, as detected by serum amylase and lipase levels as well as histologically, titres of replicating CVB4 in the pancreas were virtually unaffected. COAM treatment diminished the infection-associated MMP-9 levels and also resulted in a decreased influx of neutrophils into the infected pancreas. Moreover, we demonstrate that titres of replicating virus in the pancreas did not directly correlate with the severity of disease. In conclusion, our data suggest that immunopathological effects, rather than direct virus-induced destruction, are responsible for the damage to acinar tissue in CVB4-induced pancreatitis.