Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3- and viral-induced expression of type I interferons.

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.

L-tryptophan-kynurenine pathway metabolites regulate type I IFNs of acute viral myocarditis in mice.

The activity of IDO that catalyzes the degradation of tryptophan (Trp) into kynurenine (Kyn) increases after diseases caused by different infectious agents. Previously, we demonstrated that IDO has an important immunomodulatory function in immune-related diseases. However, the pathophysiological role of IDO following acute viral infection is not fully understood. To investigate the role of IDO in the l-Trp-Kyn pathway during acute viral myocarditis, mice were infected with encephalomyocarditis virus, which induces acute myocarditis. We used IDO-deficient (IDO(-/-)) mice and mice treated with 1-methyl-d,l-Trp (1-MT), an inhibitor of IDO, to study the importance of Trp-Kyn pathway metabolites. Postinfection with encephalomyocarditis virus infection, the serum levels of Kyn increased, whereas those of Trp decreased, and IDO activity increased in the spleen and heart. The survival rate of IDO(-/-) or 1-MT-treated mice was significantly greater than that of IDO(+/+) mice. Indeed, the viral load was suppressed in the IDO(-/-) or 1-MT-treated mice. Furthermore, the levels of type I IFNs in IDO(-/-) mice and IDO(-/-) bone marrow-transplanted IDO(+/+) mice were significantly higher than those in IDO(+/+) mice, and treatment of IDO(-/-) mice with Kyn metabolites eliminated the effects of IDO(-/-) on the improved survival rates. These results suggest that IDO has an important role in acute viral myocarditis. Specifically, IDO increases the accumulation of Kyn pathway metabolites, which suppress type I IFNs production and enhance viral replication. We concluded that inhibition of the Trp-Kyn pathway ameliorates acute viral myocarditis.

The epitope integration site for vaccine antigens determines virus control while maintaining efficacy in an engineered cancer vaccine.

Picornaviruses have been developed as potential therapies for gene delivery and vaccination. One drawback to their use is the potential for recombination and viral persistence. Therefore, the engineering strategies used must take into account the possibility for virus escape. We have developed Theiler's murine encephalomyelitis virus (TMEV) as a potential vaccine vector for use in immunotherapy. This study shows that insertion of a vaccine epitope at a unique site within the TMEV leader protein can dramatically increase the type I interferon (IFN) response to infection and promote rapid viral clearance. This live virus vaccine maintains its ability to drive antigen-specific CD8(+) T-cell responses to a model antigen as well as to the weakly immunogenic tumor antigen Her2/neu. Furthermore, the epitope integration site does not affect the efficacy of this vaccine as cancer immunotherapy for treating models of melanoma and breast cancer as demonstrated by delayed tumor outgrowth and increased survival in animals implanted with these tumors. These findings show that an attenuated virus retaining limited ability to replicate nonetheless can effectively mobilize CD8(+) cellular immunity and will be important for the design of picornavirus vectors used as immunotherapy in clinical settings.

The IRF-3/Bax-mediated apoptotic pathway, activated by viral cytoplasmic RNA and DNA, inhibits virus replication.

Induction of apoptosis in cells infected by Sendai virus (SeV), which triggers the cytosolic RIG-I pathway, requires the presence of interferon regulatory factor 3 (IRF-3). Independent of IRF-3's transcriptional role, a novel IRF-3 activation pathway causes its interaction with the proapoptotic protein Bax and its mitochondrial translocation to induce apoptosis. Here we report that two other RNA viruses, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV), may also activate the same pathway. Moreover, cytosolic DNA, produced by adenovirus or introduced by transfection, activated the pathway in an RNA polymerase III-dependent fashion. To evaluate the contribution of this newly discovered apoptotic pathway to the host's overall antiviral response, we measured the efficiencies of replication of various viruses in vitro and viral pathogenesis in vivo, using cells and mice that are selectively deficient in components required for the apoptotic pathway of IRF-3. Our results clearly demonstrate that the IRF-3/Bax-mediated apoptotic signaling branch contributes significantly to the host's protection from viral infection and consequent pathogenesis.

Antiviral effects of a transgenic RNA-dependent RNA polymerase.

Transgenic expression of the RNA-dependent RNA polymerase 3D(pol) inhibited infection of Theiler's murine encephalitis virus (TMEV), a picornavirus from which it was derived. Here, we infected 3D(pol) transgenic mice with another picornavirus, as well as an alphaherpesvirus and a rhabdovirus. 3D(pol) transgenic FVB mice had significantly lower viral loads and survived longer after infection with all three types of viruses than nontransgenic FVB mice. Viral inhibition among three different types of virus by transgenic 3D(pol) suggests that the mechanism of action is not the direct interference with picornaviral 3D(pol) but instead may be the changing of host cells to an antiviral state before or after viral infection occurs, as basal interferon levels were higher in 3D(pol) transgenic mice before infection. Further study of this mechanism may open new possibilities for future antiviral therapy.

Glucocorticoid exposure alters the pathogenesis of Theiler's murine encephalomyelitis virus during acute infection.

Previous research has shown that chronic restraint stress exacerbates Theiler's virus infection, a murine model for CNS inflammation and multiple sclerosis. The current set of experiments was designed to evaluate the potential role of glucocorticoids in the deleterious effects of restraint stress on acute CNS inflammatory disease. Exposure to chronic restraint stress resulted in elevated levels of corticosterone as well as increased clinical scores and weight loss (Experiment 1). In addition, corticosterone administration alone exacerbated behavioral signs of TMEV-induced sickness (i.e. decreased body weight, increased symptoms of encephalitis, and increased mortality) and reduced inflammation in the CNS (Experiment 2). Infected subjects receiving exogenous corticosterone showed exacerbation of acute phase measures of sickness and severe mortality as well as decreased viral clearance from CNS (Experiment 3). These findings indicate that corticosterone exposure alone is sufficient to exacerbate acute CNS inflammatory disease.

Isolation and genetic characterization of encephalomyocarditis virus 1 from a deceased captive hamadryas baboon.

In 2007, numerous hamadryas baboons (Papio hamadryas) died suddenly in an aviary of a primate institute in Sochi, Russia, in the absence of prior clinical signs. Necropsies were suggestive of encephalomyocarditis virus infection, but RT-PCR assays with commonly used primers were negative. Here we report the histopathological results obtained during necropsies and the isolation and genomic characterization of a divergent strain of encephalomyocarditis virus 1 (EMCV-1) from heart tissue of one of the succumbed hamadryas baboons. Phylogenetic analysis indicates that the isolated virus belongs to the newly proposed EMCV-1 lineage G, which clusters alongside lineage C ("Mengo virus"). This study is the first report describing a lineage G strain of EMCV-1 as the etiological agent of a lethal disease outbreak among captive nonhuman primates in Europe.

Targeted overexpression of elafin protects mice against cardiac dysfunction and mortality following viral myocarditis.

Serine elastases degrade elastin, stimulate vascular smooth muscle cell migration and proliferation, and are associated with myocardial damage. To evaluate the impact of elastase inhibition on cardiovascular development and disease, transgenic mice were created in which the mouse preproendothelin-1 promoter was used to target elafin overexpression to the cardiovascular system. To distinguish the transgene from endogenous elafin, constructs were made incorporating a FLAG sequence; the COOH-terminus FLAG-tagged elafin construct produced a stable, functionally active gene product and was used to create transgenic mice. Consistent with endothelin expression, abundant elafin mRNA was observed in transgenic F1 embryos (embryonic day 13.5) and in adult transgenic mice heart, trachea, aorta, kidney, lung, and skin, but not in liver, spleen, and intestine. Functional activity of the transgene was confirmed by heightened myocardial elastase inhibitory activity. No tissue abnormalities were detected by light microscopy or elastin content. However, injection of 10 plaque-forming units (PFU) of encephalomyocarditis virus resulted in death within 11 days in 10 out of 12 nontransgenic mice compared with one out of nine transgenic littermates. This reduced mortality was associated with better cardiac function and less myocardial inflammatory damage. Thus, elafin expression may confer a protective advantage in myocarditis and other inflammatory diseases.

Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection.

Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection.

Pimobendan inhibits the production of proinflammatory cytokines and gene expression of inducible nitric oxide synthase in a murine model of viral myocarditis.

OBJECTIVES: This study was designed to examine the effects of pimobendan in a murine model of viral myocarditis in relation to proinflammatory cytokine production and nitric oxide (NO) synthesis by inducible NO synthase (iNOS) in the heart. BACKGROUND: Pimobendan has been recently confirmed to improve both acute and chronic heart failure. Since the modulation of myocardial necrosis and contractile dysfunction by various proinflammatory cytokines may be partially mediated by the production of nitric oxide, the effects of pimobendan on the production ofproinflammatory cytokines and NO were investigated in an animal model of viral myocarditis involving heart failure. METHODS: DBA/2 mice were inoculated with the encephalomyocarditis virus. To observe its effect on survival up to 14 days, pimobendan (0.1 mg/kg or 1 mg/kg) or vehicles were given from the day of virus inoculation (day 0) orally once daily. The effects of pimobendan on histological changes, cytokine production, NO production and iNOS gene expression in the heart were studied in mice treated either with pimobendan, 1 mg/kg or with vehicles only, and sacrificed seven days after virus inoculation. RESULTS: The survival of mice improved in a dose-dependent fashion such that a significant difference (p < 0.02) was found between the higher-dose pimobendan group (20 of 30 [66.7%]) and the control group (11 of 30 [36.7%]). Histological scores for cellular infiltration (1.1+/-0.1 vs. 2.0+/-0.0, p < 0.001), intracardiac tumor necrosis factor (TNF)-alpha (18.2+/-1.8 vs. 35.8+/-4.2 pg/mg heart, p < 0.001) and interleukin (IL)-1beta (9.3 +/-1.2 vs. 26.6+/-7.1 pg/mg heart, p < 0.01) were significantly lower in the mice given pimobendan versus those of the control mice. Interleukin-6 levels (7.1+/-0.8 vs. 9.2+/-1.9 pg/mg heart) were also lower in the mice treated with pimobendan. Furthermore, intracardiac NO production was significantly (p < 0.001) less in the pimobendan group (0.165+/-0.004 nmol/mg heart) than in the control group (0.291+/-0.051 nmol/mg heart), and intracardiac iNOS gene expression in the mice given pimobendan was 74% lower than it was in the control animals (p < 0.01). CONCLUSIONS: These findings suggest that the beneficial effects of pimobendan in viral myocarditis are partially mediated by the inhibition of both proinflammatory cytokine production and NO synthesis by iNOS.

Antiviral profile of Nyctanthes arbortristis L. against encephalitis causing viruses.

The ethanolic extracts, various fractions and two pure compounds isolated from the plant N. arbortris were tested against Encephalomyocarditis Virus (EMCV) and Semliki Forest Virus (SFV). Pronounced in vitro virus inhibitory activity was observed with the ethanolic and n-butanol fractions as well as with the pure compounds arbortristoside A and arbortristoside C. In addition, ethanolic extracts and n-butanol fraction protected EMCV infected mice to the extent of 40 and 60% respectively against SFV at a daily dose of 125 mg/kg body weight.

Isolation, molecular and phylogenetic analysis of encephalomyocarditis virus strain GS01 in China.

Encephalomyocarditis virus (EMCV) is a small non-enveloped, single-stranded RNA virus. It can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. Diseases caused by EMCV currently affect the swine industry worldwide. In this study, an EMCV strain was isolated from an aborted fetus in western China. It was identified by reverse-transcription polymerase chain reaction (RT-PCR) and genome sequencing. The subsequent results showed that the virus could produce a specific cytopathic effect on BHK-21 cells and could cause severe clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that the GS01 strain was 79.9-99.9% identical with other isolates worldwide. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage 1, 2, 3, 4, and 5 based on the nucleotide sequences of the entire ORF and VP3/VP1 junction, as well as 3D gene. GS01 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain GS01 isolated from an aborted pig fetus in western China was fatal to mice and provided new epidemiologic data on EMCV in China.

Oromucosal interferon therapy: relationship between antiviral activity and viral load.

Intraperitoneal (i.p.) administration of 20,000 IU recombinant murine IFN-alpha (rMuIFN-alpha) was highly effective in protecting mice challenged i.p. with doses of encephalomyocarditis virus (EMCV) ranging from 44 to 440 LD(50) (p<0.001). Oromucosal (o.m.) IFN therapy was also found to be effective in protecting mice challenged with a lethal dose of EMCV. Thus, 40% of animals infected with 44 LD(50) of EMCV and treated o.m. with 20,000 IU rMuIFN-alpha survived infection with a mean survival time of 12.0 +/- 2.46 days relative to a mean of 6.11 +/- 0.38 days in the control group (p<0.05). Oromucosal IFN therapy was found to be ineffective, however, in animals infected with higher doses of EMCV (88-440 LD(50)), even though intraperitoneal administration of the same dose of rMuIFN-alpha resulted in the survival of 90%, 50%, and 60% of animals infected with 88, 220, and 440 LD(50) of EMCV, respectively. These results suggest that oromucosal IFN therapy is effective at relatively low viral load only and that the mechanism of action of oromucosal IFN therapy may be different from that of parenterally administered IFN. Our results suggest that oromucosal IFN therapy may be most effective in chronic viral infections as an alternative to parenterally administered IFN, which is clinically effective but poorly tolerated.

Efficacy and safety of orally/sublingually, intranasally, and intraperitoneally administered recombinant murine interferon in the treatment of murine encephalomyocarditis virus.

Interferons (IFN) have been shown to be effective in protecting animals against lethal viral infections when administered systemically in relatively high doses. Intraperitoneal (i.p.) injection of mice with encephalomyocarditis virus (EMCV) gives rise to a rapidly progressive fatal disease characterized by central nervous system involvement and encephalitis. IFN-alpha has been shown to be effective in protecting mice against lethal EMCV infection when given via parenteral and oral/sublingual routes. The current study was designed to explore the ability of orally/sublingually and intranasally (i.n.) administered IFN-alpha to treat mice infected with EMCV in support of a planned clinical trial to evaluate efficacy of oral IFN-alpha in human viral infections. The primary objective of the study was to determine the efficacy of recombinant murine IFN-alpha (rMuIFN-alpha) in the treatment of mice infected with 100 LD(50) EMCV following oral, i.n., and i.p. administration at doses of 20,000 and 100,000 IU. The results of the current experiment did not indicate protection from infection with EMCV in mice that received IFN by the i.n. or oral/sublingual routes. The negative controls, infection of mice with 100 LD(50) of EMCV followed by treatment with excipient via all three routes, resulted in death of nearly all mice, as expected. The positive control, treatment of EMCV-infected (100 LD(50)) mice with rMuIFN-alpha via the i.p. route, was successful in protecting a significant number of mice from death compared with matched controls. This study points out the need to determine the optimum conditions for administration of oral/sublingual or i.n. IFN to insure maximum efficacy against viral infections.

Antiviral action of trehalose dimycolate against EMC virus: role of macrophages and interferon alpha/beta.

Preventive treatment of mice with trehalose 6,6' dimycolate (TDM), an immunomodulator of bacterial origin, enhances their resistance to encephalomyocarditis (EMC) virus infection. The protective effect of TDM is totally abolished by the injection of silica particles in mice, demonstrating the role of macrophages in the antiviral action of TDM. In vitro, peritoneal macrophages from mice treated with TDM (TDM-PM) exhibit an intrinsic antiviral activity against EMC virus, while resident peritoneal macrophages (RES-PM) are permissive to this virus. Greater amounts of interferon are detected in supernatants of cultures of TDM-PM than of RES-PM. Neutralization of interferon (IFN) by addition in vitro of anti-IFN alpha/beta serum markedly reduces the antiviral activity of TDM-PM. These results indicate that interferon alpha/beta is involved in the intrinsic anti-EMC virus activity of peritoneal macrophages from mice treated with TDM.

Cardioviral poly(C) tracts and viral pathogenesis.

Mengovirus is a prototypical member of the cardiovirus genus of the family Picornaviridae. The positive-strand RNA genome is 7761 bases in length and encodes a polyprotein of 2293 amino acids. The 5' non-coding region (758 bases) contains an unusual homopolymeric poly(C) tract, which in the wild-type virus, has a sequence of C50UC10. We have discovered through genetic engineering that truncation or deletion of this poly(C) sequence yields infectious virus isolates that grow well in cell culture, but are 10(6) to 10(9) fold less pathogenic to mice than the wild type strain. Animals receiving sublethal doses of the short poly(C) strains characteristically develop high levels of neutralizing antibodies and acquire lifelong protective immunity against challenge with wild type virus. Effectively, the genetically engineered strains are superb vaccines against cardiovirus disease. Moreover, their potential is not limited to murine hosts. Pigs and sub-human primates have also been protectively vaccinated with short poly(C) tract Mengoviruses. The molecular mechanism of poly(C)-mediated pathogenesis is currently under study. Most hypotheses link the activity to induction of the antiviral cytokine, interferon.

Embryonic stem cells attenuate viral myocarditis in murine model.

We used mice to test our hypothesis that in response to viral invasion, stem cells may migrate into the heart and attenuate the effect of viral myocarditis. Male BALB/c mice were divided into three groups: mouse embryonic stem (ES) cell control, encephalomyocarditis virus (EMCV), and EMCV + ES cells. After administration of ES cells via tail vein, mice were immediately inoculated with EMCV. Mice were sacrificed at different days after EMCV inoculation. Mortality was recorded. Inflammatory cell infiltration and necrosis (major pathological changes of viral myocarditis) were evaluated by hematoxylin-eosin staining. ES cell migration and differentiation were identified by immunofluorescence. The survival rate in the EMCV + ES cell group (80%) was significantly increased (p < 0.05) over the EMCV-alone group (64%). Also, the incidence of inflammatory cell infiltration and myocardial lesions was lower in the EMCV + ES cell mice. Furthermore, the result of green fluorescent protein (GFP) and alpha-actinin analysis indicated that ES cells migrated into the heart and differentiated into myocytes after virus inoculation. In conclusion, ES cells significantly increased the survival of viral myocarditis mice and also decreased the necrosis and infiltration of inflammatory cells. These results demonstrated the ability of stem cells to mitigate the effects of viral infection on the heart and illustrated their potential therapeutic application to other mammalian species, including humans.

Heat stress aggravates viral myocarditis in mice.

While a beneficial effect of hyperthermia on viral infection has been hypothesized, there are no data on viral myocarditis in vivo. To investigate whether hyperthermia might attenuate the course or severity of viral myocarditis, we studied the pathological changes in a murine model of viral myocarditis. C3H mice were inoculated i.p. with the encephalomyocarditis virus (500 pfu). They were anesthetized and heated to a body temperature of 42.5+/-0.2 degrees C for 30 min. The latter was performed 4 hr before (n=28, HB) or 4 hr after (n=28, HA) the viral inoculation; results were compared with nonheated, infected controls (n=30, Cont). Cardiac viral titers were recorded on day 3, and the body weight (BW), heart weight (HW) and pathological changes were recorded on days 5 and 10. The incidence of spontaneous mortality on day 10 was significantly higher in the HA group (all deaths occurring by day 7 post-inoculation) as compared with the HB (35%) or Cont (18%) groups. Viral titers in the HA group (n=4) were significantly (P<0.05) higher than those in the Cont (n=7) or HB (n=7) groups (4.11+/-0.54 vs 3.01+/-0.44 and 3.23+/-0.45 LogTCID50/mg, respectively). On day 5, the HW, the BW/HW ratio, and the severity of myocardial necrosis were all significantly higher in the HA than in the Cont and HB groups. To confirm the effect of hyperthermia on the expression of heart shock protein (HSP), immunohistochemical staining was done in the virus-infected hearts. The nucleus and cytoplasm of the injured myocardium in the HA group strongly expressed HSP70, whereas the HB and Cont groups were negative for this protein. In conclusion, induction of hyperthermia after viral inoculation aggravated the viral-induced myocardial necrosis and increased the mortality rate in a murine model of viral myocarditis and induced myocardial heat shock protein 70.

Encephalomyocarditis (EMC) virus-induced testicular lesion in BALB/c mice.

Characteristics of encephalomyocarditis (EMC) virus-induced testicular lesions were investigated in 4- and 8-week-old BALB/c male mice after intraperitoneal (i.p.) and intratesticular (left) (i.t.) inoculation of the D variant of EMC virus (EMC-D). Apart from variation in severity and incidence, the histopathological nature of the resultant testicular lesion was similar in all infected mice, and was characterized by degeneration and necrosis of germinal cells and spermatogonia with inflammatory infiltration. Almost all the inoculated left testes of the i.t. group developed marked lesions. In general, the virus titre in the testis and incidence of testicular lesions were higher in 4-week-old mice than in 8-week-old mice. In addition, testicular lesions developed earlier and with a higher incidence in the PBS-inoculated right testis of the i.t. group than in either testis of the i.p. group of the same age.

Encephalomyocarditis virus infections in an Australian zoo.

Fatal encephalomyocarditis virus (EMCV) infections in a ring-tailed lemur (Lemur catta), a squirrel monkey (Saimiri sciureus), three mandrills (Mandrillus sphinx), a chimpanzee (Pan troglodytes), a pygmy hippopotamus (Choeropsis liberiensis), and two Goodfellows tree kangaroos (Dendrolagus goodfellowi) occurred at Taronga Zoo. This is the first description of EMCV in a zoological collection outside of the United States. Regardless of species, the most common clinical presentation was sudden death. The gross pathologic changes were diffuse or focal pallor of the myocardium with occasional marked pulmonary congestion. Necrotizing nonsuppurative myocarditis was consistently present. EMCV was isolated from only one of 54 feral rodents examined. No antibodies to EMCV were detected with a serum neutralization test in 79 stored sera from a wide variety of zoo mammals. Titers of 1:16, 1:16, and 1:4 were recorded for a spider monkey (Aeteles geoffroyi), a lion (Panthera leo), and an orangutan (Pongo pygmaeus), respectively. Of seven mandrills tested in 1988, six had measurable virus titers. Later testing indicated that these titers did not persist, and one mandrill with a titer > 1:128 in 1988 subsequently succumbed to EMCV infection in 1991.