CVB3 Infection Research Articles

Abstract 18847: Visualization of Inflammation and Characterization of Lesions in Viral Myocarditis Mouse Model by Combined 19F-MR Cell Tracking and 1H Cardiac MRI

Introduction Myocarditis is a challenging disease for diagnosis and treatment. Recently, we have established a non-invasive cellular 19F-MR method to visualize inflammation of myocarditis in mice [1]. In this study, we correlated detection of inflammation by 19F-MR and tissue characterization by 1H-MR in viral myocarditis mouse model. Methods Viral myocarditis was induced by infecting ABY mice (n=5 infected+3 control) with CVB3. 19F-perfluorocarbon emulsion (PFC) (150μl) was injected i.v. 13 days post-infection to label monocytes and macrophages in blood. The labeling efficiency was examined by FACS in blood samples. 19F/1H-images were acquired at a 7T MR. 19F-MRI was superimposed on 1H-Cine to localize inflammation in myocardium. Lesions were characterized by T2WI, T2*WI and Gd-DTPA enhancement. MR findings were examined by Histology and immunohistochemistry (IHC). Results Blood FACS and confocal microscopy on IHC revealed that the 19F-MR signal in myocardium was due to internalized PFC in macrophages. 19F-MR detected macrophage infiltrating myocarditis lesions. Edema and Gd enhancement could be found in some lesions. Interestingly, most patchy 19F signal co-localized with T2* hypo-intense signal in lesions, which has been reported in various mouse strains post CVB3 infection [2]. Necrosis, T cells, fibrosis, and mineral deposition were found in lesions by histology and IHC. Conclusion 19F-MRI unambiguously detected active recruitment of macrophages to myocarditis lesions. On T2*WI signal voids in lesions were closely related to 19F signal in location and volume, which indicates its association to inflammation. Caution should be taken to apply iron oxide particles to visualizing inflammation in viral myocarditis, while the significance and mechanism of this contrast, especially its existence in human, need further investigation. 1. Ye et al ISMRM proceeding 2012. 2. Helluy et al. ISMRM proceeding 2011. This work was supported partly by BMBF 01EZ0816.

Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20μg YW002 per mouse. When adjusted the amount at 10μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.

Impaired cardiac microvascular endothelial cells function induced by Coxsackievirus B3 infection and its potential role in cardiac fibrosis

CVB3 virus tropism and tissue access are modulated by cardiac microvascular endothelial cells (CMVECs) in the context of microvasculature. This study was designed to examine biological behaviors of CMVECs following CVB3 infection and its possible effects on cardiac remodeling. Data demonstrated that CVB3 increased caspase-3 activities, Bax/Bcl-2 protein ratio and TGF-β1 levels in CMVECs, accompanying with elevated microvascular permeability. Double immunofluorescence revealed co-localization of endothelial markers (CD31 and VE-cadherin) and mesenchymal markers (FSP1 and αSMA) in infected CMVECs. Western blot demonstrated that CVB3 significantly decreased the expression of endothelial markers and increased the expression of mesenchymal markers, which were reversed by SB431542 (inhibitor of TGF-β1), indicating that endothelial-to-mesenchymal transition following CVB3 infection was probably induced by CMVECs-derived TGF-β1. Excess extracellular matrix was produced by myocardial cells incubated with supernatants of infected CMVECs. Our results displayed that CVB3 induced notable biological changes of CMVECs, which may contribute to cardiac fibrosis.

Cytokine and Chemokine Production by Human Pancreatic Islets Upon Enterovirus Infection

Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-α) as well as various chemotactic proteins, such as IFN-γ–induced protein 10, macrophage inflammatory protein (MIP)-1α, MIP-1β, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet–inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis.

Stabilin‐1 protects against adverse inflammation and injury during viral myocarditis

Stabilin‐1 is a transmembrane glycoprotein expressed on activated macrophages and endothelial cells of new capillaries during tissue remodeling. In the coxsackie B3 (CVB3) murine model of viral myocarditis, stabilin‐1 expression increased significantly and was primarily expressed in macrophages. This was also seen in endomyocardial biopsies from patients with fulminant myocarditis, where stabilin‐1 was expressed by CD‐68+ macrophages, forming a protective barrier around the healthy tissue. When challenged with the CVB3 virus, Stabilin‐1 null mice showed increased cardiac related mortality as compared to wild‐type mice (p<0,001, n=19). Histological analysis showed that the stabilin‐1 null mice had increased cardiac necrosis due to CD3+ T‐lymphocyte infiltration, and lower F4/80+ macrophages as compared to their wild‐type littermates, suggesting that stabilin‐1 may be important for monocytes recruitment. Thioglycolate induced peritonitis in stabilin‐1 WT and null mice also resulted in fewer monocytes recruited. Depletion of circulating monocytes with clodronate liposomes in wild‐type mice induced a similar phenotype after CVB3 infection. We identify stabilin‐1 as an important upregulated protein during VM in both mice and humans and that absence of stabilin‐1 results in increased cardiac inflammation, injury and mortality, demonstrating a novel cardio‐protective effect of stabilin‐1.

Differential expression of coxsackievirus and adenovirus receptor on alveolar epithelial cells between fetal and adult mice determines their different susceptibility to coxsackievirus B infection

Coxsackievirus B (CVB) can cause aseptic meningitis, myocarditis and respiratory disease, especially in newborn infants. To compare the susceptibility to CVB infection of fetal and adult mice, we prepared primary alveolar epithelial cells (AECs) from lungs of BALB/c mice. In contrast to fetal mouse AECs, those of adults were less susceptible to CVB3 infection, as indicated by decreased cytopathic effects, and reduced levels of viral particles bound at the cell surface. In adult mouse AECs, amplification of the viral genome and virus capsid protein VP1 synthesis were concomitantly reduced. In addition, the cell-surface expression of coxsackievirus and adenovirus receptor (CAR), which plays a key role in the initiation of CVB and pulmonary infection, was downregulated in adult mouse AECs. These findings demonstrate that adult mouse AECs are less susceptible to CVB3 due to decreased CAR levels. Thus, these findings strongly indicate that the level of virus receptors on AECs is one of the crucial determinants for the age-dependence of CVB virulence in the mouse lung.

Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X(L) and Bax protein levels were observed upon CVB3 infection during 5-24h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.

Quantitative Trait Locus Analysis, Pathway Analysis, and Consomic Mapping Show Genetic Variants of Tnni3k, Fpgt, or H28 Control Susceptibility to Viral Myocarditis

Coxsackievirus B3 (CVB3) infection is the most common cause of viral myocarditis. The pathogenesis of viral myocarditis is strongly controlled by host genetic factors. Although certain indispensable components of immunity have been identified, the genes and pathways underlying natural variation between individuals remain unclear. Previously, we isolated the viral myocarditis susceptibility 1 (Vms1) locus on chromosome 3, which influences pathogenesis. We hypothesized that confirmation and further study of Vms1 controlling CVB3-mediated pathology, combined with pathway analysis and consomic mapping approaches, would elucidate both pathological and protective mechanisms accounting for natural variation in response to CVB3 infection. Vms1 was originally mapped to chromosome 3 using a segregating cross between susceptible A/J and resistant B10.A mice. To validate Vms1, C57BL/6J-Chr 3(A)/NaJ (a chromosome substitution strain that carries a diploid A/J chromosome 3) were used to replicate susceptibility compared with resistant C57BL/6J (B6). A second segregating F2 cross was generated between these, confirming both the localization and effects of Vms1. Microarray analysis of the four strains (A/J, B10.A, C57BL/6J, and C57BL/6J-Chr 3(A)/NaJ) illuminated a core program of response to CVB3 in all strains that is comprised mainly of IFN-stimulated genes. Microarray analysis also revealed strain-specific differential expression programs and genes that may be prognostic or diagnostic of susceptibility to CVB3 infection. A combination of analyses revealed very strong evidence for the existence and location of Vms1. Differentially expressed pathways were identified by microarray, and candidate gene analysis revealed Fpgt, H28, and Tnni3k as likely candidates for Vms1.

Effects of Yiqi Huoxue Recipe (益气活血方) and Coxsackie virus B type 3 on the expression of ribosomal protein S20 in rat cardiac myocytes

To study the effects of yiqi huoxue recipe and Coxsackie B virus type 3 (CVB3) on the expression of ribosomal protein S20 in rat cardiac myocytes, to explore the pathogenesis of myocarditis induced by CVB3 and the mechanism of yiqi huoxue recipe on gene level, and to further investigate whether yiqi huoxue recipe is an effective prescription for CVB3 myocarditis. A modified suppression subtractive hybridization (SSH) was used to isolate differentially expressed genes between the CVB3 infection group and the treatment group with yiqi huoxue recipe. The results were further verified by fluorescence RT-PCR. The results of SSH showed that the gene expression of ribosomal protein S20 in the treatment group was higher than that in the CVB3 infection group (P<0.05), which agreed with the results of fluorescent RT-PCR. Down-regulation of ribosomal protein S20 mRNA expression might be one of the mechanisms in CVB3 myocarditis, and yiqi huoxue recipe could achieve the treatment of viral myocarditis by regulating the expression of ribosomal protein S20.

Galectin-9 administration ameliorates CVB3 induced myocarditis by promoting the proliferation of regulatory T cells and alternatively activated Th2 cells

In this study we explored the effects of galectin-9 on CVB3 induced myocarditis and its possible mechanisms involved. We demonstrated that galectin-9 expression was significantly up-regulated in the myocardium following CVB3 infection and was correlated with the severity of viral myocarditis. To explore whether galectin-9 may have therapeutic effect on the CVB3 induced myocarditis, galectin-9 was administered daily to mice following CVB3 infection. Significantly reduced CD4(+) T cells and remarkably increased regulatory T cells frequency in the heart tissue were found as compared to the non-treated mice. It was accompanied by a significant decreased level of Th1 cytokines as TNF-α and IFN-γ both in the myocardium and serum, and an increased level of Th2 cytokines such as IL-4 and IL-10. Galectin-9 was further found to promote the proliferation of regulatory T cells and elevated IL-4-secreting Th2 cells. It may represent as a novel therapeutic strategy in treating Th1-mediated inflammatory cardiac disease.

Effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 in CVB3 infection model in rat cardiac myocytes

Objective To study the effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 (MRPL51) in CVB3 infection model in rat cardiac myocytes, to reveal the pathogenesis of CVB3 myocarditis in the genetic level, to explore the therapeutic mechanism of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis, and to confirm the validity of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis. Methods After establishing CVB3 infection model and treatment model with recipe for activating blood circulation and nourishing qi by culturing neonatal rat myocardial cells, a modified suppression subtractive hybridization (SSH) was used to isolate differentially expressed genes between two model groups. These results were further verified by fluorescence RT-PCR. Results The results of SSH showed that gene expression of the treatment group was lower than that of the CVB3 infection group. The results of fluorescent RT-PCR which agreed with that of SSH displayed the threshold cycle number (Ct) in the treatment group was higher than the virus group. Conclusion Up-regulation of MRPL51 might be one of the pathogenesis of CVB3 myocarditis. The recipe for activating blood circulation and nourishing qi could treat viral myocarditis by regulating the expression of MRPL51. Key words: Viral myocarditis ; Recipe for activating blood circulation and nourishing qi ; Suppression subtractive hybridization (SSH) ; Mitochondrial ribosomal protein L51 (MRPL51) ;

Proteasome Activator REGγ Enhances Coxsackieviral Infection by Facilitating p53 Degradation

Coxsackievirus B3 (CVB3) is a small RNA virus associated with diseases such as myocarditis, meningitis, and pancreatitis. We have previously demonstrated that proteasome inhibition reduces CVB3 replication and attenuates virus-induced myocarditis. However, the underlying mechanisms by which the ubiquitin/proteasome system regulates CVB replication remain unclear. In this study, we investigated the role of REGγ, a member of the 11S proteasome activator, in CVB3 replication. We showed that overexpression of REGγ promoted CVB3 replication but that knockdown of REGγ led to reduced CVB3 replication. We further demonstrated that REGγ-mediated p53 proteolysis contributes, as least in part, to the proviral function of REGγ. Although total protein levels of REGγ remained unaltered after CVB3 infection, virus infection induced a redistribution of REGγ from the nucleus to the cytoplasm, rendering an opportunity for a direct interaction of REGγ with viral proteins and/or host proteins (e.g., p53), which controls viral growth and thereby enhances viral infectivity. Further analyses suggested a potential modification of REGγ by SUMO following CVB3 infection, which was verified by both in vitro and in vivo sumoylation assays. Sumoylation of REGγ may play a role in its nuclear export during CVB3 infection. Taken together, our results present the first evidence that the host REGγ pathway is utilized and modified during CVB3 infection to promote efficient viral replication.

Regulating Viral Myocarditis: Allografted Regulatory T Cells Decrease Immune Infiltration and Viral Load

Coxsackievirus was first discovered as a filterable agent associated with a paralytic syndrome, so named for its identification in Coxsackie, New York (coxsackievirus type A).1 Coxsackievirus type B (CVB) was isolated the following year from patients with aseptic meningitis,2 and by the mid-1950s, an association with acute myocarditis in humans was becoming clear.3–6 Many other viruses have since been shown to cause myocarditis and its long-term sequelae, arrhythmias, dilated cardiomyopathy, and heart failure. By example, adenovirus, herpesviruses, and influenza can cause myocarditis in humans and models of heart failure. Because immune cell infiltration is an easily seen feature of myocarditis, it is perhaps not surprising that the inflammatory response would be posited as a major cause of tissue injury during viral myocarditis. However, the virus itself has lytic and destructive potential, and a range of data indicate both viral and immune contributions to cardiomyocyte and interstitial damage during myocarditis. The question is often posed: Is damage primarily virus-mediated or more so the product of an overzealous immune response and autoimmunity? Perhaps the greatest error in the discussion has been approaching these 2 facets of the disease process in a mutually exclusive manner. Shi et al7 now show in this issue of Circulation that virus replication and the immune response are exquisitely intertwined in myocarditis pathogenesis. Article see p 2624 Over the last 3 decades, it has become apparent that viral load is a primary determinant of viral myocarditis severity,8–11 the virus causing extensive, early, direct injury to the myocardium through its replication. Results cited here all indicate significant injury to the myocardium before cardiac immune infiltration, supporting the view that considerable damage to the heart is directly mediated by the virus alone. An argument has been made previously for the importance of chronic virus replication …

Left ventricular enlargement in coxsackievirus-B3 induced chronic myocarditis — ongoing inflammation and an imbalance of the matrix degrading system

Enteroviruses, especially Coxsackie B3 virus (CVB-3), cause acute viral myocarditis, but the detailed mechanisms leading to chronic left ventricular dysfunction and dilatation remain elusive. Myocardial tissues of CVB-3 infected and sham infected male swr/J mice were analyzed after hemodynamic evaluation on days 4, 7, and 28 p.i. by RT-PCR, gelatin zymography, ELISA, immunohistochemistry, sirius red staining, and luxol fast blue staining. In the early phase after infection an abnormal diastolic function was the main hemodynamic finding. CVB-3 infection caused impairment of left ventricular function combined with ventricular dilatation 7 and 28 days post-infection. These hemodynamic findings were associated with relevant upregulation of different cytokines (IL-1β, IL-6, IL-10, INF-γ, and TNF-α) in the acute phase with persistent over-expression of IL-6, IL-10, and INF-γ  in the chronic phase. This virus induced myocardial inflammation was linked to a significant induced MMP/TIMP system (MMP-2,-3,-8, TIMP-1, uPA, tPA–mRNA expression, and MMP-2-activity) in the acute and chronic phase leading to imbalance in the MMP/TIMP-ratio at day 28. This imbalance in the MMP/TIMP system was significantly correlated to the development of ventricular dilatation. Viral persistence induces chronic myocardial inflammation and an imbalance of the matrix degrading system, associated with the development of left ventricular dysfunction and dilatation in chronic murine myocarditis.

Prevention of Cardiac Dysfunction in Acute Coxsackievirus B3 Cardiomyopathy by Inducible Expression of a Soluble Coxsackievirus-Adenovirus Receptor

Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach. We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59+/-3.8 versus 45.4+/-2.7 mm Hg, median 59 versus 45.8 mm Hg, P<0.01; dP/dt(max) 3645.1+/-443.6 versus 2057.9+/-490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P<0.01; and dP/dt(min) -2125.5+/-330.5 versus -1310.2+/-330.3 mm Hg/s, median -2083.7 versus -1295.9 mm Hg/s, P<0.01) and improved contractility if induced concomitantly with infection (left ventricular pressure 76.4+/-19.2 versus 56.8+/-10.3 mm Hg, median 74.8 versus 54.4 mm Hg, P<0.05; dP/dt(max) 5214.2+/-1786.2 versus 3011.6+/-918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P<0.05), respectively. Importantly, hemodynamics of animals treated with AdG12 (plus doxycycline) were similar to uninfected controls. Preinfection induction of sCAR-Fc completely blocked and concomitant induction strongly reduced cardiac CVB3 infection, myocardial injury, and inflammation. AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions.

CXCL10 Inhibits Viral Replication Through Recruitment of Natural Killer Cells in Coxsackievirus B3-Induced Myocarditis

Coxsackievirus (CV)B3 is the primary cause of viral myocarditis. We previously observed CXC chemokine ligand 10 (CXCL10) upregulation in the myocardium early in infection. However, the impact of CXCL10 in CVB3-induced myocarditis is unknown. Using isolated primary mouse cardiomyocytes we demonstrated for the first time that cardiomyocytes can express CXCL10 on interferon-gamma stimulation. To explore the role of CXCL10 in CVB3-induced myocarditis, both CXCL10 transgenic and knockout mice were used. Following CVB3 challenges, the viral titer in the hearts inversely correlated with the levels of CXCL10 at early phase of infection before visible immune infiltration. Furthermore, as compared with the control mice, the decreased virus titers in the CXCL10 transgenic mouse hearts led to less cardiac damage and better cardiac function and vice verse in the knockout mice. This antiviral ability of CXCL10 might be through recruitment of natural killer (NK) cells to the heart and increased interferon-gamma expression early in infection. At day 7 postinfection, with massive influx of mononuclear cells the expression of CXCL10 enhanced the infiltration of CXCR3(+) cells, CD4(+), and CD8(+) T cells, as well as the expression of associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines failed to alter the viral clearance and mice survival. These results suggest the protective role of CXCL10 during the early course of CVB3 infection, which is attributed to the recruitment of NK cells. Nonetheless, CXCL10-directed chemoattractant effect is not sufficient for host to clear the virus in the heart.

IFN-γ production dominates the early human natural killer cell response to Coxsackievirus infection

Coxsackieviruses (CV) are important human pathogens that have been implicated in the pathogenesis of several diseases, including myocarditis and pancreatitis. How the human immune system recognizes and controls CV infections is not well understood. Studies in mice suggest that natural killer (NK) cells play a critical role in viral clearance and host survival, but the mechanism(s) by which human NK cells may contribute to the host anti-CV defence has not been investigated. Here we show that CVB3 infection markedly reduces HLA class I cell surface expression but does not increase the expression of the activating NK cell receptor ligands MICA/B and ULBP1-3 on human cells. We also demonstrate that the lowered target cell HLA class I surface expression does not correlate with an increased susceptibility to NK cell-mediated killing. However, NK cells responded with a robust production of interferon gamma (IFN-gamma) when peripheral blood mononuclear cells were cocultured with infected cells. In summary, this study shows that CVB3 interferes with the expression of NK cell receptor ligands on infected cells and indicates that IFN-gamma production, rather than cytotoxicity, marks the early human NK cell response to CVB3 infection.

Expression of short hairpin RNAs against the coxsackievirus B3 exerts potential antiviral effects in Cos-7 cells and in mice

Chemically synthesized small interfering RNA (siRNA) has been used as an anti-coxsackievirus B3 (CVB3) agent . Herein, we investigated whether vector-derived short hairpin RNAs (shRNA) targeting CVB3 can exert antiviral activities, prior to their further application to viral vector system for efficient in vivo administration. Employing transient transfection assays to in vivo mouse models as well as to in vitro Cos-7 cell cultures, we directly demonstrated the potential antiviral activity of shRNAs following challenges with infectious CVB3. Of the six shRNAs that we designed, three prevented cell death from CVB3 infection by suppressing viral replication and viral production in Cos-7 cells. These were shRNA 2, which targeted the capsid protein VP1, and shRNAs 4 and 5, which targeted two different regions of the RNA-dependent RNA polymerase 3D. Furthermore, shRNAs 2 and 5 also exerted strong antiviral effects in viral replication in vivo, accompanied by attenuated pancreatic tissue damage. Through this direct evaluation system we addressed the development and application of vector-derived shRNAs as an anti-CVB3 agent, revealing new target sequences.

Inhibition of Coxsackievirus B3 in Cell Cultures and in Mice by Peptide-Conjugated Morpholino Oligomers Targeting the Internal Ribosome Entry Site

Coxsackievirus B3 (CVB3) is a primary cause of viral myocarditis, yet no effective therapeutic against CVB3 is available. Nucleic acid-based interventional strategies against various viruses, including CVB3, have shown promise experimentally, but limited stability and inefficient delivery in vivo remain as obstacles to their potential as therapeutics. We employed phosphorodiamidate morpholino oligomers (PMO) conjugated to a cell-penetrating arginine-rich peptide, P007 (to form PPMO), to address these issues. Eight CVB3-specific PPMO were evaluated with HeLa cells and HL-1 cardiomyocytes in culture and in a murine infection model. One of the PPMO (PPMO-6), designed to target a sequence in the 3' portion of the CVB3 internal ribosomal entry site, was found to be especially potent against CVB3. Treatment of cells with PPMO-6 prior to CVB3 infection produced an approximately 3-log(10) decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when PPMO-6 treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of PPMO-6 once prior to and once after CVB3 infection showed an approximately 2-log(10)-decreased viral titer in the myocardium at 7 days postinfection and a significantly decreased level of cardiac tissue damage, compared to the controls. Thus, PPMO-6 provided potent inhibition of CVB3 amplification both in cell cultures and in vivo and appears worthy of further evaluation as a candidate for clinical development.

Pyrrolidine Dithiocarbamate Reduces Coxsackievirus B3 Replication through Inhibition of the Ubiquitin-Proteasome Pathway

Coxsackievirus B3 (CVB3) is one of the most common pathogens for viral myocarditis. The lack of effective therapeutics for CVB3-caused viral diseases underscores the importance of searching for antiviral compounds. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and is recently reported to inhibit ubiquitin-proteasome-mediated proteolysis. Previous studies have shown that PDTC inhibits replication of rhinovirus, influenza virus, and poliovirus. In the present study, we report that PDTC is a potent inhibitor of CVB3. Coxsackievirus-infected HeLa cells treated with PDTC showed a significant reduction of CVB3 viral RNA synthesis, viral protein VP1 expression, and viral progeny release. Similar to previous observation that divalent ions mediate the function of PDTC, we further report that serum-containing copper and zinc are required for its antiviral activity. CVB3 infection resulted in massive generation of reactive oxygen species (ROS). Although PDTC alleviated ROS generation, the antiviral activity was unlikely dependent on its antioxidant effect because the potent antioxidant, N-acetyl-L-cysteine, failed to inhibit CVB3 replication. Consistent with previous reports that PDTC inhibits ubiquitin-proteasome-mediated protein degradation, we found that PDTC treatment led to the accumulation of several short-lived proteins in infected cells. We further provide evidence that the inhibitory effect of PDTC on protein degradation was not due to inhibition of proteasome activity but likely modulation of ubiquitination. Together with our previous findings that proteasome inhibition reduces CVB3 replication (H. Luo, J. Zhang, C. Cheung, A. Suarez, B. M. McManus, and D. Yang, Am. J. Pathol. 163:381-385, 2003), results in this study suggest a strong antiviral effect of PDTC on coxsackievirus, likely through inhibition of the ubiquitin-proteasome pathway.