Variant Of Encephalomyocarditis Virus Research Articles

Quantitative comparison of gene co-expression in a bicistronic vector harboring IRES or coding sequence of porcine teschovirus 2A peptide

Simultaneous expression of multiple target genes is often required in biotechnology. Multicistronic vectors coding for several proteins are being actively developed for this purpose. In commercially available vectors different variants ofencephalomyocarditis virus internal ribosome entry site (IRES EMCV) are used most often. However, many researchers consider that utilization ofself-cleaving 2A peptides sequences within multi- and bicistronic vectors is more promising. In this work, we compared the efficiency of gene expression in cells transfected with bicistronic vectors based on IRES EMCV and 2A peptide sequence derived form porcine teschovirus-1 (P2A). Efficiency ofgene expression was determined in three mammalian cell lines by measurement of co-expression levels of genes coding for RFP and EGFP proteins linked by IRES or P2A sequence. Higher level oftransgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence.

Role of Nitric Oxide in the Pathogenesis of Encephalomyocarditis Virus-Induced Diabetes in Mice

The D variant of encephalomyocarditis virus (EMC-D virus) causes diabetes in mice by destroying pancreatic beta cells. In mice infected with a low dose of EMC-D virus, macrophages play an important role in beta-cell destruction by producing soluble mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). To investigate the role of NO and inducible NO synthase (iNOS) in the development of diabetes in EMC-D virus-infected mice, we infected iNOS-deficient DBA/2 mice with EMC-D virus (2 x 10(2) PFU/mouse). Mean blood glucose levels in EMC-D virus-infected iNOS-deficient mice and wild-type mice were 205.5 and 466.7 mg/dl, respectively. Insulitis and macrophage infiltration were reduced in islets of iNOS-deficient mice compared with wild-type mice at 3 days after EMC-D virus infection. Apoptosis of beta cells was decreased in iNOS-deficient mice, as evidenced by reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. There were no differences in mRNA expression of antiapoptotic molecules Bcl-2, Bcl-xL, Bcl-w, Mcl-1, cIAP-1, and cIAP-2 between wild-type and iNOS-deficient mice, whereas expression of proapoptotic Bax and Bak mRNAs was significantly decreased in iNOS-deficient mice. Expression of IL-1beta and TNF-alpha mRNAs was significantly decreased in both islets and macrophages of iNOS-deficient mice compared with wild-type mice after EMC-D virus infection. Nuclear factor kappaB was less activated in macrophages of iNOS-deficient mice after virus infection. We conclude that NO plays an important role in the activation of macrophages and apoptosis of pancreatic beta cells in EMC-D virus-infected mice and that deficient iNOS gene expression inhibits macrophage activation and beta-cell apoptosis, contributing to prevention of EMC-D virus-induced diabetes.

Destruction of human retinoblastoma after treatment by the E variant of encephalomyocarditis virus

The oncolytic effects of encephalomyocarditis (EMC) virus were examined in human retinoblastoma cell (Y79) cultures which were infected with 10(4 )tissue culture infectious doses (TCIDs) of the E variant of EMC (EMC-E) virus. The TCIDs were used to titer the maximum effect of EMC virus on L-929 cells. In-vitro studies showed 90% cytopathic effect (CPE) at 24 h and 100% CPE at 52 h. The CPE was used to observe pathologic effects of the cells. In-vivo studies employing human retinoblastoma grown as a tumor in nude mice, revealed degeneration of 80% of the tumor cells at 3 days and total destruction at 4 days following inoculation with the EMC-E virus. The virus is highly neurotropic in mice, but is usually not pathogenic in man. These studies suggest a possible new direction in the treatment of retinoblastoma and other malignant tumors using the viral technology.

Experimental encephalomyocarditis virus infection in pregnant mice

The present study was carried out to clarify the mode of encephalomyocarditis (EMC) virus infection in pregnant mice. Pregnant BALB/c mice were intraperitoneally inoculated with the D variant of EMC virus (EMC-D) (5 × 10 2 PFU/mouse) on 11 days of gestation and killed at 1, 3, and 5 days post-inoculation (DPI). The virus titer (dam's serum, placenta, and fetus), histopathology (fetus, placenta, and uterus), distribution of viral RNA (fetus, placenta and uterus), and ultrastructure (fetal heart and placenta) were examined. No deaths occurred to fetuses at 1 DPI but almost all fetuses died at 5 DPI. The virus titers of dam's serum and placenta were elevated at 1 DPI, peaked at 3 DPI, and the former was not detected at 5 DPI. The virus titer of fetus was first elevated at 3 DPI and the level was lower than those of others. Histopathological changes and signals of viral RNAs detected by in situ hybridization (ISH) were observed in the spongiotrophoblast layer of placenta and in the fetal myocardium and liver at 3 DPI. The uterus was free from lesions and signals of viral RNA. Ultrastructural changes developed in trophoblast cells and giant cells in the spongiotrophoblast layer at and after 1 DPI and in fetal myocardial cells at 3 DPI. In the cytoplasm of trophoblast cells and giant cells, aggregations of virus-like particles 20–30 nm in diameter were observed in crystalline array. These results suggest that trophoblast cells and giant cells in the spongiotrophoblast layer are the main target of EMC virus in the placenta and that placental damage as well as the direct effect of virus to fetuses may be a cause of fetal death.

Susceptibility of primary culture neurons from rats of different ages to encephalomyocarditis (EMC) virus infection

The changes in susceptibility of neurons to the D variant of EMC virus (EMC-D) (10 6 PFU/well) were investigated in developing hippocampal primary cultures from postnatal days of 1, 7, and 56 Fischer 344 rats (P1, P7, and P56) for up to 12 h after infection (12 HAI). The virus titer of primary culture neurons increased at 1 HAI, decreased at 2 HAI, increased at 3 HAI, peaked at 8 HAI, and decreased at 12 HAI in all age groups. The titers at 1 and 8 HAI were lowest in P56 cultures. The virus titer of neurons was always higher than that of culture media, especially at 1 HAI, in P1 cultures, whereas the former was lower than the latter from 2 to 3 HAI in P7 cultures and from 2 to 4 HAI in P56 cultures, respectively. Signals of viral RNA detected by in situ hybridization were first observed in the peripheral cytoplasm of neurons at 1 HAI in P1 and P7 cultures and at 4 HAI in P56 cultures, respectively. The signals spread to a large or whole area of cytoplasm and also to processes thereafter. The number of viral RNA-positive neurons and the amount of signals decreased with age. The present results indicated that the susceptibility of primary culture neurons to EMC-D decreased with age but viral replication still occurred in P56 cultures.

Age-related changes in susceptibility of rat brain slice cultures including hippocampus to encephalomyocarditis virus.

Replication of the D variant of encephalomyocarditis virus (EMC-D) and its cytopathic effects were studied in the brain slice cultures including hippocampus (hippocampal slice) obtained from postnatal 1-, 4-, 7-, 14-, 28-and 56-day-old Fischer 344 rats. At 0, 12, 24, 36 and 48 h after infection, virus titres of the slices and culture media were assayed. Viral replication was observed in cultures from 1-to 28-day-old rats, and the highest titre was recorded in the slice and culture medium from the youngest rat. The peak of virus titre decreased with age and no distinct viral replication was observed in the cultures from 56-day-old rats. Light microscopy revealed that degenerative and necrotic changes appeared in the infected hippocampal slices from 1- to 28-day-old rats, and the changes became less prominent with age. In situ hybridization and indirect immunofluorescence staining showed that positive signals of viral RNA and antigen were prominent in younger rats and decreased with age. These results suggest that an age-related decrease in the susceptibility of rat brain to EMC-D is less related to the maturation of the immune system but possibly to that of the neurone.

Prevention of encephalomyocarditis virus-induced diabetes in mice by inhibition of the tyrosine kinase signalling pathway and subsequent suppression of nitric oxide production in macrophages.

Macrophages comprise the major population of cells infiltrating pancreatic islets during the early stages of infection in DBA/2 mice by the D variant of encephalomyocarditis virus (EMC-D virus). Inactivation of macrophages prior to viral infection almost completely prevents EMC-D virus-induced diabetes. This investigation was initiated to determine whether a tyrosine kinase signalling pathway might be involved in the activation of macrophages by EMC-D virus infection and whether tyrosine kinase inhibitors might, therefore, abrogate EMC-D virus-induced diabetes in vivo. When isolated macrophages were infected with EMC-D virus, inducible nitric oxide synthase mRNA was expressed and nitric oxide was subsequently produced. Treatment of macrophages with the tyrosine kinase inhibitor tyrphostin AG126, but not tyrphostin AG556, prior to EMC-D virus infection blocked the production of nitric oxide. The infection of macrophages with EMC-D virus also resulted in the activation of the mitogen-activated protein kinases (MAPKs) p42(MAPK/ERK2)/p44(MAPK/ERK1), p38(MAPK), and p46/p54(JNK). In accord with the greater potency of AG126 than of AG556 in blocking EMC-D virus-mediated macrophage activation, the incidence of diabetes in EMC-D virus-infected mice treated with AG126 (25%) was much lower than that in AG556-treated (75%) or vehicle-treated (88%) control mice. We conclude that EMC-D virus-induced activation of macrophages resulting in macrophage-mediated beta-cell destruction can be prevented by the inhibition of a tyrosine kinase signalling pathway involved in macrophage activation.

Encephalomyocarditis (EMC) virus infection in PC12 and C6 cells.

PC12 cells derived rom rat pheochromocytoma and C6 cells derived from rat glioma were infected with 0.3 plaque forming units (PFU)/cell of the D variant of encephalomyocarditis virus (EMC-D), after pretreatment with or without nerve growth factor (NGF). The virus titres in medium and cells were investigated at 6, 12, 24, 48 and 72 h post infection (HPI), and histopathology and viral antigens in cells were examined at 24 and 48 HPI, respectively. As a result, neither viral replication nor light and electron microscopic changes were observed in PC12 cell cultures without NGF-pretreatment. On the contrary, in PC12 cell cultures with NGF-pretreatment, the virus titre prominently increased at 12 HPI, and peaked at 48 HPI. In addition, distinct histological and ultrastructural changes with viral antigens in cells were observed. C6 cells showed similar morphology and susceptibility to EMC-D-infection irrespective of NGF-pretreatment. Namely, the virus titres in C6 cell cultures increased slightly and viral antigens were found in a small number of C6 cells, but there were no evident histological and ultrastructural changes. These results suggest that PC12 cells pretreated with NGF and C6 cells are susceptible to EMC-D infection in vitro.

Cytokines produced early in picornavirus infection reflect resistance or susceptibility to disease.

Gender bias favoring female resistance to picornavirus disease is not seen in ICR Swiss mice following infection with the MM strain of encephalomyocarditis virus (EMCV) (causing encephalitis and death) as it is with D variant of EMCV (causing diabetes in males). To define this difference, an in vitro virus-infected splenocyte culture system was used to explore virus effects on lymphoid cells. Infected and sham-infected splenocyte cultures, prepared from both genders of mice and infected with either virus variant, were examined for immunoregulatory cytokines in the first 24 h of infection using ELISA or bioassays. Disease resistance was associated with increased levels of interferon-y (IFN-gamma) and undetectable levels of interleukin-10 (IL-10) by 12 h postinfection in splenocytes from ICR Swiss females infected with EMCV-D. Disease susceptibility was associated with high levels of IL-10 at 12 h after infection of spleen cells from ICR Swiss males infected with EMCV-D or from both genders infected with EMCV-MM. This information was used to protect susceptible mice against picornavirus disease (either diabetes or death) by giving them an inducer of IFN-alpha/beta, to induce natural killer (NK)-like cells to produce high levels of IFN-gamma and rat monoclonal anti-IL-10 to neutralize the effects of mouse IL-10.

An age-related change in susceptibility of rat brain to encephalomyocarditis virus infection.

Rats were inoculated intraperitoneally (i.p.) or intracerebrally (i.c.) with 1 x 10(4) plaque forming units (PFU)/animal of the D variant of encephalomyocarditis virus (EMC-D) at 2, 4, 7, 14, 28 or 56 days of age for virological and histopathological examination. In the i.p.-inoculation study, neither viral replication nor lesions were detected in the animals inoculated at 28 and 56 days of age. In the animals inoculated when younger than 14 days of age, lesions were restricted to the brain although viral replication was detected in the brain, heart and pancreas. The brain lesions were characterized by acute meningoencephalitis with neuronal necrosis in the cerebral cortex, hippocampus and thalamus, and viral RNA was detected in degenerated and/or intact neurons. In the i.c.-inoculation study, similar age-related changes in susceptibility of rat brain to EMC-D infection were observed, but a minor difference was that viral replication and lesions were still detected in the hippocampus of some animals inoculated at 28 days of age. These results suggest that an age-related decrease in the susceptibility of rat brain to EMC virus infection may reflect an age-related change in the susceptibility of neurons themselves as well as in maturation of the immune system.

Beneficial effects of amlodipine in a murine model of congestive heart failure induced by viral myocarditis. A possible mechanism through inhibition of nitric oxide production.

Although calcium channel blockers have not been shown to be beneficial for the treatment of patients with heart failure, a recent clinical trial demonstrated a favorable effect of amlodipine on the survival of patients with heart failure resulting from nonischemic dilated cardiomyopathy. We investigated the effects of amlodipine on a murine model of congestive heart failure induced by the M variant of encephalomyocarditis virus (EMCV). Four-week-old male DBA/2 mice were inoculated with EMCV and administered amlodipine, diltiazem, or vehicle PO for 2 weeks. The heart weight-to-body weight ratio and the histopathological grades of myocardial lesions were significantly lower and survival was significantly increased in the amlodipine-treated group (P < .01, P < .05, and P < .05, respectively) than in the control group. In vitro, amlodipine added to murine J774A.1 macrophages concomitant with EMCV inhibited nitrite formation in a concentration-dependent manner, but diltiazem did not. Furthermore, NG-monomethyl-L-arginine, an inhibitor of NO synthesis, decreased myocardial lesions significantly in this murine model. Immunohistochemistry revealed that the number of cells stained with antibody against an inducible NO synthase decreased significantly in the amlodipine-treated group compared with that in the control group (P < .01). Amlodipine appears to have a protective effect against myocardial injury in this animal model of congestive heart failure. The therapeutic effect of amlodipine may be in part resulting from inhibition of overproduction of NO.

Detection of viral RNA by electron microscopic in situ hybridization (ISH-EM) in the germinal epithelium of mice infected with encephalomyocarditis (EMC) virus.

Electron microscopic in situ hybridization (ISH-EM) was first applied to the detection of viral RNA in the germinal epithelium of mice inoculated i.p. with 10(5) plaque-forming units/mouse of the D variant of encephalomyocarditis virus (EMC-D). Signals of viral RNA were first detected in a small number of Sertoli cells showing mild degeneration at 2 days post inoculation, and 2 days later, they were also detected in germinal cells and spermatogonia when Sertoli cells showed prominent degeneration. The results clearly demonstrated that the first site of viral attack in the germinal epithelium was Sertoli cell in the case of EMC-D-induced mouse orchitis.

Encephalomyocarditis (EMC) virus infection in the common vole, Microtus arvalis

Encephalomyocarditis (EMC) virus infection in the common vole was examined for the first time. Sixteen 8-week-old males inoculated intraperitoneally with 10(5) plaque-forming units (pfu)/animal of the D variant of EMC virus were killed 3 and 7 days after inoculation (3 and 7 DAI). Viral replication was detected in the brain (10(5) pfu/g), heart (10(4) pfu/g) and pancreas (10(7) pfu/g) of all 8 animals at 3DAI. It was found in the pancreas (10(3) pfu/g) of all 8 animals and in the brain (10(4) pfu/g) of 2 of 8 animals at 7 DAI. Histopathological changes were observed in the brain (mild mononuclear cell infiltration around capillaries and sporadic pyknosis of neurons), heart (minimal myocardial necrosis) and pancreas (prominent acinar cell necrosis with inflammatory exudation) at 3DAI. At 7 DAI, replacement of necrotic tissues by mesenchymal cells and regeneration of acinar cells were conspicuous in the pancreas. Throughout the experimental period, no evidence for diabetogenic effect was found.

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.

Persistence of the D variant of encephalomyocarditis virus in the ICR-Swiss mouse.

The D variant of encephalomyocarditis virus (EMCV-D) is used in the murine model to study virus-induced, acute-onset diabetes mellitus (IDDM) and myocarditis. In this model, viral replication and disease occur within seven days post infection (p.i.), and by Day 10 p.i., no infectious virus is detectable. The present study examined the possibility that EMCV-D persists in ICR-Swiss mice after the acute infection is resolved. The data show that viral antigen is detected at 28 days p.i. within the pancreatic islets of 8/10 males and 13/14 females, and within the heart valves of all animals tested. Histologic examination of the organs at 28 days p.i. suggests the development of chronic obstructive pancreatitis, and shows almost fully healed lesions in the myocardium. These observations indicate that the murine model for the study of EMCV-D induced IDDM may be extended to investigate chronic pancreatitis and heart-valve disease.

Does the gender difference in interferon production seen in picornavirus-infected spleen cell cultures from ICR Swiss mice have any in vivo significance?

Splenocyte cultures from female ICR Swiss mice produced greater interferon (IFN) levels, particularly IFN-gamma, than did cultures from males by 12 h post-infection (pi) with the D variant of encephalomyocarditis virus (EMCV-D). This early IFN-gamma is produced by natural killer (NK)-like cells and is dependent on plastic adherent cells and IFN-alpha/beta. In this study, we evaluated the significance of this observation on the innate resistance of ICR Swiss females to EMCV-D-mediated disease. Treatment of females with rabbit anti-mouse IFN-alpha/beta serum rendered them susceptible to the diabetogenicity of EMCV-D. Although sera from both sexes of ICR Swiss mice exhibited peak IFN levels day 3 pi, IFN-gamma was present in the sera of males at only 1 day pi and in the sera of females at days 1-3 pi. Females cleared virus from the circulation by day 2 pi, 1 day earlier than did males. Flow cytometric evaluations of lymphoid cell phenotypes in spleens and pancreata of infected mice revealed that percentages of L3T4+ cells were significantly decreased only in spleens from males at day 1 pi and were diminished along with Ly2+ cells in pancreata of males at 7 days pi, suggesting that T-cell responses were impaired in virus-infected males.

Pathogenesis of the B variant of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV) are immunologically indistinguishable by hyperimmune serum, but, with the exception of EMCV-B, each produces a different disease syndrome and infects the central nervous system in mice infected via the intraperitoneal route of inoculation. The B variant is benign in that it does not produce any overt signs of infection at doses as high as 10(6) pfu per animal. The present study was carried out to determine if EMCV-B was pathogenic when administered via the intracranial route and, if so, to delineate the area(s) of the brain infected. The results show that, when given i.c., EMCV-B is similar to other variants of EMCV in that it infects and replicates in the brain, causing encephalitis, neuronal necrosis in Ammon's horn of the hippocampus, and clinical signs of infection. The data indicate that receptor sites for EMCV-B are present on brain cells and suggest that its benign nature when given by the intraperitoneal route reflects an inability to cross the blood-brain barrier.

Transmissibility of the D variant of encephalomyocarditis virus (EMC-D) in mice.

Transmissibility of the D variant of encephalomyocarditis virus (EMC-D) was examined. Eight-week-old ICR:CD-1 male mice inoculated with 10(5) plaque forming units (PFU)/animal of EMC-D intranasally, orally or intraperitoneally showed marked viraemia and prominent pancreatic lesions at 2 days after inoculation (2 DAI), and excreted virus in faeces from 2 to 8 DAI (virus titre: 10(3)-10(5) PFU/g). Only a small proportion of control mice housed with EMC-D-inoculated mice for 10 days developed viraemia and pancreatic lesions.

Development of a radiolabeled nucleic acid probe for the detection of encephalomyocarditis virus of swine.

Encephalomyocarditis virus (EMCV) is a member of the genus Cardiovirus of the family Picomaviridae and infects a number of species including mice, elephants, squirrels, swine, and humans. EMCV infections in swine are usually sporadic in nature throughout the world. EMCV causes encephalitis and myocarditis with high mortality in young piglets and has also been associated with maternal reproductive disease in swine. 4,15,16,19 Transplacental infection in pregnant sows has been demonstrated, but the role of EMCV in clinical reproductive disease in the US swine herds is unclear. Serologic evidence of EMCV infection in swine without clinical problem has been reported. 22 Serologic data suggest that EMCV infections are widespread in the US swine herds, but EMCV has been isolated from only a limited number of cases. 10-14,26 Whether the poor success in EMCV isolation from aborted fetuses is due to the lability of EMCV or to the lack of any role of EMCV in swine reproductive disease has not been resolved. The presence of blocking antibodies may also interfere with the isolation of EMCV from clinical specimens. The recent isolation of porcine reproductive and respiratory syndrome (PRRS) virus from herds with reproductive failure has further confused what role EMCV has in swine reproductive failure in the US swine herds. EMCV isolates are closely related antigenically and there is probably only 1 serotype of EMCV. Variants of EMCV have been isolated that differ in organ tropism and pathogenicity. 1 Antigenic and genomic variability among various EMCV strains has also been reported and may be associated with the diversity in pathogenicity. 1,7,11,24,24 VP1 is the major polypeptide of EMCV against which virus neutralizing antibodies are directed. The VP1 gene is also the least conserved capsid gene among different members of Picornavirus. In contrast, the VP3 and VP4 genes and the 5' nontranslated region of EMCV are more conserved. 13,14,20,26 The major objective of the present study was to determine whether a nucleic acid probe specific for EMCV infection in swine could be developed. Such a probe would be useful in determining the prevalence and role of EMCV, if any, in swine reproductive failure. The EMCV strains used for this study were NVSL-FL, MN-25, ATCC VR-129B, Georgia, OVL-5/90, and Mengo. The NVSL-FL strain was isolated initially from pigs in Florida. The MN-25 strain was isolated from an aborted swine fetus obtained from a Minnesota farm that had a history of acute reproductive failure and neonatal losses. The ATCC VR-129B was originally isolated from a chimpanzee

Differences in the Two-Dimension-Gel Electrophoresis Protein Patterns of the Lethal K and Benign B Variants of Encephalomyocarditis Virus

Variants of encephalomyocarditis virus (EMCV) are indistinguishable by hyperimmune serum. In spite of their antigenic similarity, they produce different disease syndromes in susceptible strains of mice. To understand the basis for the diversity in pathogenicity, studies have been initiated to characterize each of the virus variants. In this study, two-dimensional gel electrophoresis was used to compare the proteins produced by the benign EMCV-B with those produced by lethal EMCV-K. The data show that (i) the replication cycle of each of the virus variants is characteristic of picornaviruses, (ii) the VP1 of EMCV-K is more basic than that of EMCV-B, and (iii) three proteins, one a major component of VP1, the other two with molecular weight of about 12,000, are present in EMCV-K but not in EMCV-B.