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Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

Bats are potential natural hosts of Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV). Bats appear to have some unique features in their innate immune system that inhibit viral replication causing limited clinical symptoms, and thus, contributing to the virus spill over to humans. Here, kidney epithelial cell lines derived from four bat species (Pteropus dasymallus, Rousettus leschenaultii, Rhinolophus ferrumequinum, and Miniopterus fuliginosus) and two non-bat species (Homo sapiens and Mesocricetus auratus) were infected with EMCV and JEV. The replication of EMCV and JEV was lower in the bat cell lines derived from R. leschenaultii, R. ferrumequinum, and M. fuliginosus with a higher expression level of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferon-beta (IFN-β) than that in the non-bat cell lines and a bat cell line derived from P. dasymallus. The knockdown of TLR3, RIG-I, and MDA5 in Rhinolophus bat cell line using antisense RNA oligonucleotide led to decrease IFN-β expression and increased viral replication. These results suggest that TLR3, RIG-I, and MDA5 are important for antiviral response against EMCV and JEV in Rhinolophus bats., Highlights • Some bat cell lines showed a limited cytopathic effect after EMCV and JEV infection. • EMCV and JEV replication was inhibited in some bat cell lines. • Expression of PRRs and IFN-β was highly induced in some bat cell lines. • PRRs and IFN-β contributed to antiviral response against EMCV and JEV in bats.

Background Encephalomyocarditis virus (EMCV) infection can cause reproductive failure in sows and acute myocarditis and sudden death in piglets. It has caused huge economic losses to the global pig industry and that is why it is necessary to develop effective new treatment compounds. Zedoary turmeric oil has been used for treating myocarditis. Curcumol extracted from the roots of curcuma is one of the main active ingredient of zedoary turmeric oil. The anti-EMCV activity of curcumol along with the molecular mechanisms involved with a focus on IFN-β signaling pathway was investigated in this study. Method 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the maximum non-toxic concentration (MNTC), 50% cytotoxic concentration (CC50), maximum inhibition rate (MIR) and 50% effective concentration (EC50) against EMCV. Through EMCV load, the anti-viral effect of curcumol was quantitatively determined using real-time quantitative PCR (qPCR). The effect of curcumol on the expression of IFN-β was investigated using real-time quantitative PCR and ELISA. Western blot was used to determine the amounts of MDA5, MAVS, TANK, IRF3 and P-IRF3 proteins in human embryonic kidney 293 T (HEK-293 T) cells infected with EMCV. Results The results of MTT showed that compared with the ribavirin positive control group, the maximum inhibition ratio (MIR) of curcumol was greater but the selection index (SI) value was much smaller than that of ribavirin. The results of qPCR showed that curcumol and ribavirin significantly reduced the replication of EMCV in HEK-293 T cells. The curcumol (0.025 mg/mL) treatment has significantly increased IFN-β mRNA expression in the EMCV-infected HEK-293 T cells while ribavirin treatment did not. The results of ELISA showed that curcumol (0.025 mg/mL and 0.0125 mg/mL) has significantly increased the expression of IFN-β protein in EMCV-infected HEK-293 T cells. The results of Western blot showed that curcumol can inhibit the degradation of TANK protein mediated by EMCV and promote the expression of MDA5 and P-IRF3, while the protein expression level of MAVS and IRF3 remain unchanged. Conclusion Curcumol has biological activity against EMCV which we suggest that IFN-β signaling pathway is one of its mechanisms.

Cardioviruses cause diseases in many animals including, in rare cases, humans. Although they share common features with all picornaviruses, cardioviruses have unique properties that distinguish them from other family members, including enteroviruses. One feature shared by all picornaviruses is the covalent attachment of VPg to the 5' end of genomic RNA via a phosphotyrosyl linkage. For enteroviruses, this linkage is cleaved by a host cell protein, TDP2. Since TDP2 is divergently required during enterovirus infections, we determined if TDP2 is necessary during infection by the prototype cardiovirus, EMCV. We found that EMCV yields are reduced in the absence of TDP2. We observed a decrease in viral protein accumulation and viral RNA replication in the absence of TDP2. In contrast to enterovirus infections, we found that TDP2 is modified at peak times of EMCV infection. This finding suggests a unique mechanism for cardioviruses to regulate TDP2 activity during infection.

Positive-strand RNA viruses modulate lipid homeostasis to generate unique, membranous “replication organelles” (ROs) where viral genome replication takes place. Hepatitis C virus, encephalomyocarditis virus (EMCV), and enteroviruses have convergently evolved to hijack host phosphatidylinositol 4-kinases (PI4Ks), which produce PI4P lipids, to recruit oxysterol-binding protein (OSBP), a PI4P-binding protein that shuttles cholesterol to ROs. Consistent with the proposed coupling between PI4K and OSBP, enterovirus mutants resistant to PI4KB inhibitors are also resistant to OSBP inhibitors. Here, we show that EMCV can replicate without accumulating PI4P/cholesterol at ROs, by acquiring point mutations in nonstructural protein 3A. Remarkably, the mutations conferred resistance to PI4K but not OSBP inhibitors, thereby uncoupling the levels of dependency of EMCV RNA replication on PI4K and OSBP. This work may contribute to a deeper understanding of the roles of PI4K/PI4P and OSBP/cholesterol in membrane modifications induced by positive-strand RNA viruses., Positive-strand RNA [(+)RNA] viruses are true masters of reprogramming host lipid trafficking and synthesis to support virus genome replication. Via their membrane-associated 3A protein, picornaviruses of the genus Enterovirus (e.g., poliovirus, coxsackievirus, and rhinovirus) subvert Golgi complex-localized phosphatidylinositol 4-kinase IIIβ (PI4KB) to generate “replication organelles” (ROs) enriched in phosphatidylinositol 4-phosphate (PI4P). PI4P lipids serve to accumulate oxysterol-binding protein (OSBP), which subsequently transfers cholesterol to the ROs in a PI4P-dependent manner. Single-point mutations in 3A render enteroviruses resistant to both PI4KB and OSBP inhibition, indicating coupled dependency on these host factors. Recently, we showed that encephalomyocarditis virus (EMCV), a picornavirus that belongs to the Cardiovirus genus, also builds PI4P/cholesterol-enriched ROs. Like the hepatitis C virus (HCV) of the Flaviviridae family, it does so by hijacking the endoplasmic reticulum (ER)-localized phosphatidylinositol 4-kinase IIIα (PI4KA). Here we provide genetic evidence for the critical involvement of EMCV protein 3A in this process. Using a genetic screening approach, we selected EMCV mutants with single amino acid substitutions in 3A, which rescued RNA virus replication upon small interfering RNA (siRNA) knockdown or pharmacological inhibition of PI4KA. In the presence of PI4KA inhibitors, the mutants no longer induced PI4P, OSBP, or cholesterol accumulation at ROs, which aggregated into large cytoplasmic clusters. In contrast to the enterovirus escape mutants, we observed little if any cross-resistance of EMCV mutants to OSBP inhibitors, indicating an uncoupled level of dependency of their RNA replication on PI4KA and OSBP activities. This report may contribute to a better understanding of the roles of PI4KA and OSBP in membrane modifications induced by (+)RNA viruses. IMPORTANCE Positive-strand RNA viruses modulate lipid homeostasis to generate unique, membranous “replication organelles” (ROs) where viral genome replication takes place. Hepatitis C virus, encephalomyocarditis virus (EMCV), and enteroviruses have convergently evolved to hijack host phosphatidylinositol 4-kinases (PI4Ks), which produce PI4P lipids, to recruit oxysterol-binding protein (OSBP), a PI4P-binding protein that shuttles cholesterol to ROs. Consistent with the proposed coupling between PI4K and OSBP, enterovirus mutants resistant to PI4KB inhibitors are also resistant to OSBP inhibitors. Here, we show that EMCV can replicate without accumulating PI4P/cholesterol at ROs, by acquiring point mutations in nonstructural protein 3A. Remarkably, the mutations conferred resistance to PI4K but not OSBP inhibitors, thereby uncoupling the levels of dependency of EMCV RNA replication on PI4K and OSBP. This work may contribute to a deeper understanding of the roles of PI4K/PI4P and OSBP/cholesterol in membrane modifications induced by positive-strand RNA viruses.

Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild-type Noxa restored normal cytopathic responses. Noxa regulation by virus mirrored its regulation by proteasome inhibitors or ER stress inducers and the ER stress response inhibitor salubrinal protected cells against viral cytopathic effects. Noxa mRNA and protein were synergistically upregulated by IFN or dsRNA when combined with ER stress inducers, leading to Noxa/Mcl-1 interaction, activation of Bax and pro-apoptotic caspases, degradation of Mcl-1, loss of mitochondrial membrane potential and initiation of apoptosis. These data highlight the importance of ER stress in augmenting the expression of Noxa following viral infection.

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

Encephalomyocarditis virus (EMCV) has been found on pig farms worldwide and can cause myocarditis in young pigs and reproduction disorders in sows. So far, clinical signs of EMCV have not been reported in the Netherlands. The aim of this study was to estimate the seroprevalence of EMCV infection in Dutch sow herds. A total of 277 Dutch sow herds were randomly selected, from which 3237 serum samples were collected. These samples were tested for EMCV antibodies using the virus neutralization test (VN test). The apparent prevalence of EMCV antibodies was 9.3% in the total sow population, and the apparent herd prevalence was 58.8%. An exact determination of the prevalence of EMCV infections in the Dutch sow population was not possible because the characteristics of the VN test under field circumstances were not known. However, Dutch sow herds seem to be infected with EMCV because the distribution of positive blood samples in the tested sow population was significantly different from that expected if random false-positive reactions had occurred.

Graphical abstract Novel nucleosides were designed and synthesized by [3+2] cycloaddition, and showed moderate antiviral activities. The nucleosides were tested against 12 different viruses., This paper describes a simple method for synthesizing a small library of 5-isoxazol-5-yl-2′-deoxyuridines from 5-iodo-2′-deoxyuridine. Nitrile oxides were generated in situ from oximes using a commercial bleaching agent; their cycloaddition with 5-ethynyl-2′-deoxyuridine yielded isoxazoles possessing activity against herpes simplex viruses 1 and 2, Encephalomyocarditis virus, Coxsackie B3, and vesicular stomatitis virus; these isoxazoles were, however, inactive against corona virus, influenza virus, and HIV.

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001, eLife digest A virus is basically molecules of DNA or RNA inside a protein shell, and in order to reproduce, it must infect a living cell and take control of it. However, the attacked cell will fight back and try to eliminate the invader. Activation of this so-called innate immune response requires the host cells to recognize that they have been infected, which they do by detecting the tell-tale molecules that indicate the presence of the virus. When an RNA virus infects a cell, the tell-tale molecules are often atypical RNA molecules carried by the virus or produced as the virus replicates. Recognition of this ‘foreign’ material by receptor proteins inside the cell triggers the production of molecules called interferons, which activate the innate defence systems that eliminate the virus. Different receptor proteins recognize different RNA viruses. For example, a receptor called MDA5 is known to respond to the picornaviruses, some of which can cause inflammation of the brain and heart muscle. However, the identities of the specific RNA molecules that are recognized by the MDA5 receptor have not been known. Deddouche et al. have now identified one such RNA molecule with the help of a second receptor protein, called LGP2. The LGP2 receptor is not able to give the signal to produce interferons, so it is thought to work by binding to the RNA molecule to form a complex that is then relayed to MDA5 to give this signal. By isolating the complexes of LGP2 receptor from picornavirus-infected cells and sequencing the associated RNA, it was possible to identify the mystery RNA trigger. Deddouche et al. then tested picornaviruses in which this piece of RNA had been deleted from the genome, and found that the mutant viruses triggered a much weaker interferon response. By providing insight into the ways that some viruses are detected by the innate immune system, this research may inform future work on the development of new treatments to control viral infection. DOI: http://dx.doi.org/10.7554/eLife.01535.002

PML and nuclear bodies (NBs) are implicated in antiviral defense. Indeed, our team showed that overexpression of PMLIII confers resistance to vesicular stomatitis virus, influenza virus, foamy virus but not to encephalomyocarditis virus (EMCV). I have shown during my thesis that EMCV counteracts the antiviral effect of PMLIII by inducing its degradation in SUMO and proteasome-dependent way. However, cells derived from PML knockout mice are more susceptible to EMCV infection than wild-type cells. To determine the isoforme of PML implicated in this antiviral effect, I analysed the effect of the seven PML isoforms (PMLI-PMLVII) and I showed that only stable expression of PMLIV confers resistance to EMCV by sequestring the viral polymérase 3Dpol in PML Nbs. In addition, depletion of PMLIV boosted EMCV production in interferon-treated cells. These finding sindicate the mechanism by which PML confers resistance to EMCV and reveal a new pathway mediating the antiviral activity of interferon against EMCV., PML et les corps nucléaires (CN) sont impliqués dans la défense antivirale. En effet, notre équipe a montré que la surexpression de PMLIII confère la résistance au virus de la stomatite vésiculaire, au virus de l'influenza, au virus foamy mais pas au virus de l’encéphalomyocardite (EMCV). J’ai montré dans mon travail de thèse que l’EMCV contrecarre le pouvoir antiviral de PMLIII en induisant sa dégradation par un processus dépendant du protéasome et de SUMO. Cependant, les cellules de souris invalidées pour PML sont plus sensibles à l’infection par l’EMCV que les cellules issues de souris parentales. Pour déterminer l’isoforme de PML responsable de cet effet antiviral, j’ai analysé l’effet des sept isoformes de PML (PMLI-VII) et j’ai montré que seule l’expression en stable de PMLIV confère la résistance à l’EMCV en séquestrant la polymérase virale 3Dpol au sein des CN PML. De plus la déplétion de PMLIV augmente la production de l’EMCV dans les cellules traitées par l’interféron. Ces données indiquent le mécanisme par lequel PML confère la résistance à l’EMCV et révèlent que PML est l’une des protéines médiatrices des effets anti-EMCV de l’interféron.

International audience; The encephalomyocarditis virus (EMCV) is a small non-enveloped single- strand RNA virus, the causative agent of not only myocarditis and encephalitis, but also neurological diseases, reproductive disorders and diabetes in many mammalian species. EMCV pathogenesis appears to be viral strain-and host-specific, and a better understanding of EMCV virulence factors is increasingly required. Indeed, EMCV is often used as a model for diabetes and viral myocarditis, and is also widely used in immunology as a double-stranded RNA stimulus in the study of Toll-like as well as cytosolic receptors. However, EMCV virulence and properties have often been neglected. Moreover, EMCV is able to infect humans albeit with a low morbidity. Progress on xenografts, such as pig heart transplantation in humans, has raised safety concerns that need to be explored. In this review we will highlight the biology of EMCV and all known and potential virulence factors.

Background In this study, porcine encephalomyocarditis virus (EMCV) virus-like particles (VLPs) were generated using a baculovirus expression system and were tested for immunogenicity and protective efficacy in vivo. Results VLPs were successfully generated from Sf9 cells infected with recombinant baculovirus and were confirmed to be approximately 30-40 nm by transmission electron microscopy (TEM). Immunization of mice with 0.5 μg crude protein containing the VLPs resulted in significant protection from EMCV infection (90%). In swine, increased neutralizing antibody titers were observed following twice immunization with 2.0 μg crude protein containing VLPs. In addition, high levels of neutralizing antibodies (from 64 to 512 fold) were maintained during a test period following the second immunization. No severe injection site reactions were observed after immunization and all swine were healthy during the immunization period Conclusion Recombinant EMCV VLPs could represent a new vaccine candidate to protect against EMCV infection in pig farms.

Encephalomyocarditis virus (EMCV) has been found on pig farms worldwide and can cause myocarditis in young pigs and reproduction disorders in sows. So far, clinical signs of EMCV have not been reported in the Netherlands. The aim of this study was to estimate the seroprevalence of EMCV infection in Dutch sow herds. A total of 277 Dutch sow herds were randomly selected, from which 3237 serum samples were collected. These samples were tested for EMCV antibodies using the virus neutralization test (VN test). The apparent prevalence of EMCV antibodies was 9.3% in the total sow population, and the apparent herd prevalence was 58.8%. An exact determination of the prevalence of EMCV infections in the Dutch sow population was not possible because the characteristics of the VN test under field circumstances were not known. However, Dutch sow herds seem to be infected with EMCV because the distribution of positive blood samples in the tested sow population was significantly different from that expected if random false-positive reactions had occurred. In een onderzoek voor het bepalen van seroprevalentie van het Encephalomyocarditis virus op Nederlandse zeugenbedrijven werden 3237 bloedmonsters van 277 zeugenbedrijven onderzocht op antilichamen tegen dit virus m.b.v de Virus-Neutralisatietest

Treatment of cells with interferons (IFNs) induces resistance to virus infection. The 2′-5′oligo A (2-5A) synthetase/RNase L is one of the pathways leading to translation inhibition induced by IFN treatment. A murine cDNA encoding the 43-kDa 2-5A synthetase was cloned and sequenced. NIH-3T3 cell clones transfected with this cDNA expressed the enzymatic activity to various extents and exhibited resistance to encephalomyocarditis virus (EMCV) but not to vesicular stomatitis virus replication. The specific resistance to EMCV can be attributed to 2-5A synthetase.