A recent view about encephalomyocarditis virus circulating in compartmentalised animal population in Northern Italy.

Encephalomyocarditis virus (Picornaviridae, Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis and reproductive disorders in various mammals, especially in swine. Despite the disease occurred endemically in pig farms since 1997, the recent increase of death experimented in Northern Italy prompted to furtherly investigate the evolution of the virus and the actual spread of the infection. Italian EMC viruses, collected between 2013 and 2019, showed an overall antigenic stability. The in-house ELISA Monoclonal Antibodies based, able to reveal changes in seven different antigenic sites, showed only sporadic and occasional mutations in considered samples and the subsequent phylogenetic analysis confirmed antigenic panel's remarks. All the isolates could be classified within a unique lineage, which comprise other European strains and confirm that the viruses currently circulating in Italy developed from a unique common ancestor. Despite the demonstrated stability of virus, some putative newly emerged variants were detected through antigenic profile analysis and phylogenesis. Finally, the serosurvey proved that spread of EMCV is greater than the diffusion of fatal infections would suggest, due to subclinical circulation of EMCV. It demonstrated an increase in the proportion of seropositive farms, if compared with previous data with no remarkable differences between farms with and without clinical evidence of disease.

DDX56 antagonizes IFN-ß production to enhance EMCV replication by inhibiting IRF3 nuclear translocation.

DEAD (Asp-Glu-Ala-Asp)-box RNA helicases (DDX) play important roles in viral infection, either as cytosolic viral nucleic acids sensors or as essential host factors for viral replication. In this study, we identified DDX56 as a positive regulator for encephalomyocarditis virus (EMCV) replication. EMCV infection promotes DDX56 expression via its viral proteins, VP3 and 3C. We showed that DDX56 overexpression promotes EMCV replication whereas its loss dampened EMCV replication. Consequently, knockdown of DDX56 increases type I interferon (IFN) expression during EMCV infection. We also showed that DDX56 interrupts IFN regulatory factor 3 (IRF3) phosphorylation and its nucleus translocation by directly targeting KPNA3 and KPNA4 in an EMCV-triggered MDA5 signaling activation cascade leading to the blockade of IFN-ß production. Overall, we showed that DDX56 is a novel negative regulator of EMCV-mediated IFN-ß responses and that DDX56 plays a critical role in EMCV replication. These findings reveal a novel strategy for EMCV to utilize a host factor to evade the host innate immune response and provide us new insight into the function of DDX56.

C-type lectin receptor DCIR contributes to hippocampal injury in acute neurotropic virus infection.

Neurotropic viruses target the brain and contribute to neurologic diseases. C-type lectin receptors (CLRs) are pattern recognition receptors that recognize carbohydrate structures on endogenous molecules and pathogens. The myeloid CLR dendritic cell immunoreceptor (DCIR) is expressed by antigen presenting cells and mediates inhibitory intracellular signalling. To investigate the effect of DCIR on neurotropic virus infection, mice were infected experimentally with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue of TMEV-infected C57BL/6 mice and DCIR-/- mice were analysed by histology, immunohistochemistry and RT-qPCR, and spleen tissue by flow cytometry. To determine the impact of DCIR deficiency on T cell responses upon TMEV infection in vitro, antigen presentation assays were utilised. Genetic DCIR ablation in C57BL/6 mice was associated with an ameliorated hippocampal integrity together with reduced cerebral cytokine responses and reduced TMEV loads in the brain. Additionally, absence of DCIR favoured increased peripheral cytotoxic CD8+ T cell responses following TMEV infection. Co-culture experiments revealed that DCIR deficiency enhances the activation of antigen-specific CD8+ T cells by virus-exposed dendritic cells (DCs), indicated by increased release of interleukin-2 and interferon-γ. Results suggest that DCIR deficiency has a supportive influence on antiviral immune mechanisms, facilitating virus control in the brain and ameliorates neuropathology during acute neurotropic virus infection.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

Resilience in Long-Term Viral Infection: Genetic Determinants and Interactions.

Virus-induced neurological sequelae resulting from infection by Theiler's murine encephalomyelitis virus (TMEV) are used for studying human conditions ranging from epileptic seizures to demyelinating disease. Mouse strains are typically considered susceptible or resistant to TMEV infection based on viral persistence and extreme phenotypes, such as demyelination. We have identified a broader spectrum of phenotypic outcomes by infecting strains of the genetically diverse Collaborative Cross (CC) mouse resource. We evaluated the chronic-infection gene expression profiles of hippocampi and thoracic spinal cords for 19 CC strains in relation to phenotypic severity and TMEV persistence. Strains were clustered based on similar phenotypic profiles and TMEV levels at 90 days post-infection, and we categorized distinct TMEV response profiles. The three most common profiles included "resistant" and "susceptible," as before, as well as a "resilient" TMEV response group which experienced both TMEV persistence and mild neurological phenotypes even at 90 days post-infection. Each profile had a distinct gene expression signature, allowing the identification of pathways and networks specific to each TMEV response group. CC founder haplotypes for genes involved in these pathways/networks revealed candidate response-specific alleles. These alleles demonstrated pleiotropy and epigenetic (miRNA) regulation in long-term TMEV infection, with particular relevance for resilient mouse strains.

The CSF1R-Microglia Axis Has Protective Host-Specific Roles During Neurotropic Picornavirus Infection.

Viral encephalitis is a major cause of morbidity and mortality, but the manifestation of disease varies greatly between individuals even in response to the same virus. Microglia are professional antigen presenting cells that reside in the central nervous system (CNS) parenchyma that are poised to respond to viral insults. However, the role of microglia in initiating and coordinating the antiviral response is not completely understood. Utilizing Theiler's murine encephalomyelitis virus (TMEV), a neurotropic picornavirus, and PLX5622, a small molecule inhibitor of colony-stimulating factor 1 receptor (CSF1R) signaling that can deplete microglia in the CNS; we investigated the role of the CSF1R-microglia axis in neurotropic picornavirus infection of C57BL/6J and SJL/J mice. These mouse strains differ in their ability to clear TMEV and exhibit different neurological disease in response to TMEV infection. CSF1R antagonism in C57BL/6J mice, which normally clear TMEV in the CNS, led to acute fatal encephalitis. In contrast, CSF1R antagonism in SJL/J mice, which normally develop a chronic CNS TMEV infection, did not result in acute encephalitis, but exacerbated TMEV-induced demyelination. Immunologically, inhibition of CSF1R in C57BL/6J mice reduced major histocompatibility complex II expression in microglia, decreased the proportion of regulatory T cells in the CNS, and upregulated proinflammatory pathways in CNS T cells. Acute CSF1R inhibition in SJL/J mice had no effect on microglial MHC-II expression and upregulated anti-inflammatory pathways in CNS T cells, however chronic CSF1R inhibition resulted in broad immunosuppression. Our results demonstrate strain-specific effects of the CSF1R-microglia axis in the context of neurotropic viral infection as well as inherent differences in microglial antigen presentation and subsequent T cell crosstalk that contribute to susceptibility to neurotropic picornavirus infection.

Curcumol inhibits encephalomyocarditis virus by promoting IFN-ß secretion.

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-ß 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-ß 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-ß 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-ß 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-ß signaling pathway is one of its mechanisms.

Tamoxifen Application Is Associated with Transiently Increased Loss of Hippocampal Neurons following Virus Infection.

Tamoxifen is frequently used in murine knockout systems with CreER/LoxP. Besides possible neuroprotective effects, tamoxifen is described as having a negative impact on adult neurogenesis. The present study investigated the effect of a high-dose tamoxifen application on Theiler's murine encephalomyelitis virus (TMEV)-induced hippocampal damage. Two weeks after TMEV infection, 42% of the untreated TMEV-infected mice were affected by marked inflammation with neuronal loss, whereas 58% exhibited minor inflammation without neuronal loss. Irrespective of the presence of neuronal loss, untreated mice lacked TMEV antigen expression within the hippocampus at 14 days post-infection (dpi). Interestingly, tamoxifen application 0, 2 and 4, or 5, 7 and 9 dpi decelerated virus elimination and markedly increased neuronal loss to 94%, associated with increased reactive astrogliosis at 14 dpi. T cell infiltration, microgliosis and expression of water channels were similar within the inflammatory lesions, regardless of tamoxifen application. Applied at 0, 2 and 4 dpi, tamoxifen had a negative impact on the number of doublecortin (DCX)-positive cells within the dentate gyrus (DG) at 14 dpi, without a long-lasting effect on neuronal loss at 147 dpi. Thus, tamoxifen application during a TMEV infection is associated with transiently increased neuronal loss in the hippocampus, increased reactive astrogliosis and decreased neurogenesis in the DG.

IRF7-Associated Immunophenotypes Have Dichotomous Responses to Virus/Allergen Coexposure and OM-85-Induced Reprogramming.

High risk for virus-induced asthma exacerbations in children is associated with an IRF7lo immunophenotype, but the underlying mechanisms are unclear. Here, we applied a Systems Biology approach to an animal model comprising rat strains manifesting high (BN) versus low susceptibility (PVG) to experimental asthma, induced by virus/allergen coexposure, to elucidate the mechanism(s)-of-action of the high-risk asthma immunophenotype. We also investigated potential risk mitigation via pretreatment with the immune training agent OM-85. Virus/allergen coexposure in low-risk PVG rats resulted in rapid and transient airways inflammation alongside IRF7 gene network formation. In contrast, responses in high-risk BN rats were characterized by severe airways eosinophilia and exaggerated proinflammatory responses that failed to resolve, and complete absence of IRF7 gene networks. OM-85 had more profound effects in high-risk BN rats, inducing immune-related gene expression changes in lung at baseline and reducing exaggerated airway inflammatory responses to virus/allergen coexposure. In low-risk PVG rats, OM-85 boosted IRF7 gene networks in the lung but did not alter baseline gene expression or cellular influx. Distinct IRF7-associated asthma risk immunophenotypes have dichotomous responses to virus/allergen coexposure and respond differentially to OM-85 pretreatment. Extrapolating to humans, our findings suggest that the beneficial effects OM-85 pretreatment may preferentially target those in high-risk subgroups.

Transcriptome analysis following neurotropic virus infection reveals faulty innate immunity and delayed antigen presentation in mice susceptible to virus-induced demyelination.

Viral infections of the central nervous system cause acute or delayed neuropathology and clinical consequences ranging from asymptomatic courses to chronic, debilitating diseases. The outcome of viral encephalitis is partially determined by genetically programed immune response patterns of the host. Experimental infection of mice with Theiler's murine encephalomyelitis virus (TMEV) causes diverse neurologic diseases, including TMEV-induced demyelinating disease (TMEV-IDD), depending on the used mouse strain. The aim of the present study was to compare initial transcriptomic changes occurring in the brain of TMEV-infected SJL (TMEV-IDD susceptible) and C57BL/6 (TMEV-IDD resistant) mice. Animals were infected with TMEV and sacrificed 4, 7, or 14 days post infection. RNA was isolated from brain tissue and analyzed by whole-transcriptome sequencing. Selected differences were confirmed on a protein level by immunohistochemistry. In mock-infected SJL and C57BL/6 mice, >200 differentially expressed genes (DEGs) were detected. Following TMEV-infection, the number of DEGs increased to >700. Infected C57BL/6 mice showed a higher expression of transcripts related to antigen presentation via major histocompatibility complex (MHC) I, innate antiviral immune responses and cytotoxicity, compared with infected SJL animals. Expression of many of those genes was weaker or delayed in SJL mice, associated with a failure of viral clearance in this mouse strain. SJL mice showed prolonged elevation of MHC II and chemotactic genes compared with C57BL/6 mice, which presumably facilitates the induction of chronic demyelinating disease. In addition, elevated expression of several genes associated with immunomodulatory or -suppressive functions was observed in SJL mice. The exploratory study confirms previous observations in the model and provides an extensive list of new immunologic parameters potentially contributing to different outcomes of viral encephalitis in two mouse strains.

Excessive Innate Immunity Steers Pathogenic Adaptive Immunity in the Development of Theiler's Virus-Induced Demyelinating Disease.

Several virus-induced models were used to study the underlying mechanisms of multiple sclerosis (MS). The infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) establishes persistent viral infections and induces chronic inflammatory demyelinating disease. In this review, the innate and adaptive immune responses to TMEV are discussed to better understand the pathogenic mechanisms of viral infections. Professional (dendritic cells (DCs), macrophages, and B cells) and non-professional (microglia, astrocytes, and oligodendrocytes) antigen-presenting cells (APCs) are the major cell populations permissive to viral infection and involved in cytokine production. The levels of viral loads and cytokine production in the APCs correspond to the degrees of susceptibility of the mice to the TMEV-induced demyelinating diseases. TMEV infection leads to the activation of cytokine production via TLRs and MDA-5 coupled with NF-κB activation, which is required for TMEV replication. These activation signals further amplify the cytokine production and viral loads, promote the differentiation of pathogenic Th17 responses, and prevent cellular apoptosis, enabling viral persistence. Among the many chemokines and cytokines induced after viral infection, IFN α/ß plays an essential role in the downstream expression of costimulatory molecules in APCs. The excessive levels of cytokine production after viral infection facilitate the pathogenesis of TMEV-induced demyelinating disease. In particular, IL-6 and IL-1ß play critical roles in the development of pathogenic Th17 responses to viral antigens and autoantigens. These cytokines, together with TLR2, may preferentially generate deficient FoxP3+CD25- regulatory cells converting to Th17. These cytokines also inhibit the apoptosis of TMEV-infected cells and cytolytic function of CD8+ T lymphocytes (CTLs) and prolong the survival of B cells reactive to viral and self-antigens, which preferentially stimulate Th17 responses.

Double-edged effects of tamoxifen-in-oil-gavage on an infectious murine model for multiple sclerosis.

Tamoxifen gavage is a commonly used method to induce genetic modifications in cre-loxP systems. As a selective estrogen receptor modulator (SERM), the compound is known to have immunomodulatory and neuroprotective properties in non-infectious central nervous system (CNS) disorders. It can even cause complete prevention of lesion development as seen in experimental autoimmune encephalitis (EAE). The effect on infectious brain disorders is scarcely investigated. In this study, susceptible SJL mice were infected intracerebrally with Theiler's murine encephalomyelitis virus (TMEV) and treated three times with a tamoxifen-in-oil-gavage (TOG), resembling an application scheme for genetically modified mice, starting at 0, 18, or 38 days post infection (dpi). All mice developed 'TMEV-induced demyelinating disease' (TMEV-IDD) resulting in inflammation, axonal loss, and demyelination of the spinal cord. TOG had a positive effect on the numbers of oligodendrocytes and oligodendrocyte progenitor cells, irrespective of the time point of application, whereas late application (starting 38 dpi) was associated with increased demyelination of the spinal cord white matter 85 dpi. Furthermore, TOG had differential effects on the CD4+ and CD8+ T cell infiltration into the CNS, especially a long lasting increase of CD8+ cells was detected in the inflamed spinal cord, depending of the time point of TOG application. Number of TMEV-positive cells, astrogliosis, astrocyte phenotype, apoptosis, clinical score, and motor function were not measurably affected. These data indicate that tamoxifen gavage has a double-edged effect on TMEV-IDD with the promotion of oligodendrocyte differentiation and proliferation, but also increased demyelination, depending on the time point of application. The data of this study suggest that tamoxifen has also partially protective functions in infectious CNS disease. These effects should be considered in experimental studies using the cre-loxP system, especially in models investigating neuropathologies.

The Intestinal Microbiome Primes Host Innate Immunity against Enteric Virus Systemic Infection through Type I Interferon.

Intestinal microbiomes are of vital importance in antagonizing systemic viral infection. However, very little literature has shown whether commensal bacteria play a crucial role in protecting against enteric virus systemic infection from the aspect of modulating host innate immunity. In the present study, we utilized an enteric virus, encephalomyocarditis virus (EMCV), to inoculate mice treated with phosphate-buffered saline (PBS) or given an antibiotic cocktail (Abx) orally or intraperitoneally to examine the impact of microbiota depletion on virulence and viral replication in vivo Microbiota depletion exacerbated the mortality, neuropathogenesis, viremia, and viral burden in brains following EMCV infection. Furthermore, Abx-treated mice exhibited severely diminished mononuclear phagocyte activation and impaired type I interferon (IFN) production and expression of IFN-stimulated genes (ISG) in peripheral blood mononuclear cells (PBMC), spleens, and brains. With the help of fecal bacterial 16S rRNA sequencing of PBS- and Abx-treated mice, we identified a single commensal bacterium, Blautia coccoides, that can restore mononuclear phagocyte- and IFNAR (IFN-α/ß receptor)-dependent type I IFN responses to restrict systemic enteric virus infection. These findings may provide insight into the development of novel therapeutics for preventing enteric virus infection or possibly alleviating clinical diseases by activating host systemic innate immune responses via respective probiotic treatment using B. coccoidesIMPORTANCE While cumulative data indicate that indigenous commensal bacteria can facilitate enteric virus infection, little is known regarding whether intestinal microbes have a protective role in antagonizing enteric systemic infection by modulating host innate immunity. Although accumulating literature has pointed out that the microbiota has a fundamental impact on host systemic antiviral innate immune responses mediated by type I interferon (IFN), only a few specific commensal bacteria species have been revealed to be capable of regulating IFN-I and ISG expression, not to mention the underlying mechanisms. Thus, it is important to understand the cross talk between microbiota and host anti-enteric virus innate immune responses and characterize the specific bacterial species that possess protective functions. Our study demonstrates how fundamental innate immune mediators such as mononuclear phagocytes and type I IFN are regulated by commensal bacteria to antagonize enteric virus systemic infection. In particular, we have identified a novel commensal bacterium, Blautia coccoides, that can restrict enteric virus replication and neuropathogenesis by activating IFN-I and ISG responses in mononuclear phagocytes via an IFNAR- and STAT1-mediated signaling pathway.

USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS.

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.

PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs.

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR, plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on residues Thr446 and Thr451. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding or dimerization, suggesting a model where negative charges occurring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes.

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.

Saffold virus infection in elderly people with acute gastroenteritis in Sweden.

Viral gastroenteritis is a major source of morbidity and mortality, predominantly caused by so-called NOROAD viruses (norovirus, rotavirus, and adenovirus). In approximately onethird of all cases, however, the exact etiology is unknown. The in 2007 discovered human cardiovirus Saffold virus (SAFV) may prove to be a plausible candidate to explain this diagnostic gap. This virus, a member of the Picornaviridae family which is closely related to the murine viruses Theiler's murine encephalomyelitis virus and Theravirus, is a widespread pathogen and causes infection early in life. Screening of 238 fecal or vomitus samples obtained from NOROAD-negative, elderly patients with acute gastroenteritis at the University Hospital of Linköping showed that SAFV is present in low abundance (4.6%). Phylogenetic analysis of the VP1 gene revealed a Swedish isolate belonging to the highly common and in Europe widespread SAFV-3 genotype. This genotype is also related to previously reported Asian strains. This study describes the first molecular typing of a Swedish SAFV isolate and is the first report to document the circulation of SAFV among elderly people. The pathogenicity of SAFV is, as of yet, still under debate; further studies are necessary to determine its role in the development of disease.

Molecular characterization of encephalomyocarditis virus strains isolated from an African elephant and rats in a French zoo.

In November 2013, a fatal encephalomyocarditis virus (EMCV) case in a captive African elephant (Loxodonta africana) occurred at the Réserve Africaine de Sigean, a zoo in the south of France. Here we report the molecular characterization of the EMCV strains isolated from samples collected from the dead elephant and from 3 rats (Rattus rattus) captured in the zoo at the same time. The EMCV infection was confirmed by reverse-transcription real-time PCR (RT-rtPCR) and genome sequencing. Complete genome sequencing and sequence alignment indicated that the elephant's EMCV strain was 98.1-99.9% identical to the rat EMCV isolates at the nucleotide sequence level. Phylogenetic analysis of the ORF, P1, VP1, and 3D sequences revealed that the elephant and rat strains clustered into lineage A of the EMCV 1 group. To our knowledge, molecular characterization of EMCV in France and Europe has not been reported previously in a captive elephant. The full genome analyses of EMCV isolated from an elephant and rats in the same outbreak emphasizes the role of rodents in EMCV introduction and circulation in zoos.

Encephalomyocarditis Virus Abrogates the Interferon Beta Signaling Pathway via Its Structural Protein VP2.

Type I interferon (IFN)-mediated antiviral responses are critical for modulating host-virus responses, and indeed, viruses have evolved strategies to antagonize this pathway. Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen, which causes myocarditis, encephalitis, neurological disease, reproductive disorders, and diabetes in pigs. This study aims to understand how EMCV interacts with the IFN pathway. EMCV circumvents the type I IFN response by expressing proteins that antagonize cellular innate immunity. Here, we show that EMCV VP2 is a negative regulator of the IFN-ß pathway. This occurs via the degradation of the MDA5-mediated cytoplasmic double-stranded RNA (dsRNA) antiviral sensing RIG-I-like receptor (RLR) pathway. We show that structural protein VP2 of EMCV interacts with MDA5, MAVS, and TBK1 through its C terminus. In addition, we found that EMCV VP2 could significantly degrade RLRs by the proteasomal and lysosomal pathways. For the first time, EMCV VP2 was shown to play an important role in EMCV evasion of the type I IFN signaling pathway. This study expands our understanding that EMCV utilizes its capsid protein VP2 to evade the host antiviral response.IMPORTANCE Encephalomyocarditis virus is an important pathogen that can cause encephalitis, myocarditis, neurological diseases, and reproductive disorders. It also causes huge economic losses for the swine industry worldwide. Innate immunity plays an important role in defending the host from pathogen infection. Understanding pathogen microorganisms evading the host immune system is of great importance. Currently, whether EMCV evades cytosolic RNA sensing and signaling is still poorly understood. In the present study, we found that viral protein VP2 antagonized the RLR signaling pathway by degrading MDA5, MAVS, and TBK1 protein expression to facilitate viral replication in HEK293 cells. The findings in this study identify a new mechanism for EMCV evading the host's innate immune response, which provide new insights into the virus-host interaction and help develop new antiviral approaches against EMCV.

NUDT21 Links Mitochondrial IPS-1 to RLR-Containing Stress Granules and Activates Host Antiviral Defense.

Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid-inducible protein-I-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms, including type I IFN induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. In this study, we show that NUDT21, an RNA-binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of viral dsRNA. We found that despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection in both human HeLa cells and mouse macrophage cell line RAW264.7 cells. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.