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.

Interference by a non-interferon-inducing subvariant of benign encephalomyocarditis virus-B with the ability of EMCV-D to produce insulin-dependent diabetes mellitus in ICR Swiss male mice.

The D variant of encephalomyocarditis virus (EMCV-D) produces a disease syndrome that mimics insulin-dependent diabetes mellitus (IDDM) in certain mouse strains. Benign EMCV-B interferes with the ability of EMCV-D to produce IDDM. Because EMCV-B induces the production of relatively large amounts of interferon (IFN), it has been hypothesized that the interference by EMCV-B with the pathogenesis of EMCV-D is due to IFN. However, we have previously reported that in outbred ICR Swiss and inbred BALB/cByJ mice, interference by EMCV-B with the development of IDDM in response to infection with EMCV-D does not appear to involve IFN. We have isolated a subvariant of EMCV-B (EMCV-B1) which, preliminary experiments indicate, does not induce the production of detectable levels of IFN in cell culture. Studies were initiated using this subvariant to determine more conclusively if IFN is involved in interference by EMCV-B with the pathogenesis of EMCV-D. The data in the present study show that EMCV-B1 does not induce the production of detectable levels of IFN either in cell culture or in mice, but retains other reported characteristics of the parent EMCV-B, including the ability to interfere with the production of IDDM by EMCV-D in ICR Swiss male mice. These observations strengthen the hypothesis that protection of pancreatic beta cells in ICR Swiss mice by EMCV-B occurs by a mechanism other than IFN.

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.

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.

The role of interferon in the protection of ICR Swiss male mice by the nondiabetogenic variant of encephalomyocarditis virus against virus-induced diabetes mellitus.

The production of murine diabetes mellitus by the diabetogenic variant of encephalomyocarditis virus (EMCV-D) is prevented in mice preinfected for 24 h with nondiabetogenic virus (EMCV-B). It has been suggested that the protection of the animals is due to the induction of interferon (IFN) by EMCV-B. The present study was done to investigate further the role of IFN in the protection of male ICR Swiss mice against the diabetogenic effects of EMCV-D virus. The results show that this mouse strain is protected only by viable EMCV-B given by either the i.v. or i.p. route of inoculation and that the protection is not abrogated by anti-IFN gamma-globulin. Further, the animals were not protected by either IFN-alpha, IFN- beta, or IFN- alpha/beta at concentrations shown previously to protect mice against the lethal effects of other variants of EMCV. The data provide further evidence that, in the ICR Swiss mouse, IFN does not play a major role in protecting pancreatic beta-cells against infection by EMCV-D.

Differing attachment of diabetogenic and nondiabetogenic variants of encephalomyocarditis virus to beta-cells.

The D variant of encephalomyocarditis (EMC-D) virus does not induce the production of interferon (IFN) and produces an insulin-dependent diabetes mellitus (IDDM)-like syndrome in certain mouse strains. In contrast, the B variant (EMC-B) virus, which is serologically identical to EMC-D virus, is a good inducer of IFN and is nondiabetogenic. It has been postulated that IFN may play a major role in determining the ability of these two viruses to infect pancreatic beta-cells. However, recent studies have shown that ICR Swiss and BALB/cByJ male mice are not protected by IFN against EMC-D virus-induced IDDM. Furthermore, treatment of these two strains of mice with anti-IFN gamma-globulin before infection with EMC-B virus does not result in diabetes. These observations suggest that mechanisms other than the IFN system are involved in determining the ability of the viruses to infect and destroy beta-cells. Studies were initiated to identify other mechanisms of action. In this communication, we show that up to six times more EMC-D than EMC-B virus attaches to primary beta-cells extracted from male ICR Swiss mice. This difference in ability to attach to beta-cells may account for the difference in the diabetic potential of this mouse strain.

Tropism and histopathology of the D, B, K, and MM variants of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV), which are immunologically indistinguishable by hyperimmune serum, produce different disease syndromes in mice. For instance, in ICR Swiss male mice, EMCV-B produces no overt illness, EMCV-MM produces severe neurological signs followed by death, EMCV-D destroys pancreatic beta cells producing a disease syndrome resembling insulin-dependent diabetes mellitus, and EMCV-K is lethal but produces no overt signs of infection. The present study was done to determine the tissue tropism and histopathology of each of these EMCV variants in the ICR Swiss mouse model. The data show the highest concentrations in the following organs: EMCV-D in the pancreas, EMCV-B in the pancreas, EMCV-MM in the cerebrum, and EMCV-K in the medulla/brainstem. They also show that the pathological lesions produced by each variant correlate well with viral titers.

Effect of interferons and poly(I):poly(C) on the pathogenesis of the diabetogenic variant of encephalomyocarditis virus in different mouse strains.

Interferon (IFN) can either prevent or exacerbate the pathogenic effects of the diabetogenic variant of encephalomyocarditis (EMC-D) virus. The effect seen is dependent upon the mouse strain and the time of IFN administration. For example, IFN-alpha beta protects SWR/J but not ICR Swiss male mice against the diabetogenic effects of the virus. Administration of either IFN-alpha beta or the IFN-inducer poly(I):poly(C) 4 days post infection, results in an exacerbation of the infection in ICR Swiss and C57BL/6 male mice. Studies have been initiated to investigate the role of the IFN system in the pathogenesis of this virus infection. In this study IFNs or poly(I):poly(C) were administered to several mouse strains at 24 h before or 4 days after infection with EMC-D virus. The results of such treatment ranged from complete protection of the animals from the diabetogenic effects of the virus to exacerbation of the infection as reflected by the virus content in selected organs. The effect was dependent upon the mouse strain, the type of IFN, and the time of its administration in relation to virus infection.

Virus-induced murine diabetes. Enhancement by immunosuppression.

Adult, male ICR Swiss mice are susceptible to the diabetogenic effects of the D-variant of encephalomyocarditis virus (EMC-D) in contrast to adult C3H/HeJ male mice, which are relatively resistant. To date, experimental evidence suggests that the immune system plays a role in the pathogenesis of this infection. We have investigated the potential involvement of the immune system in the pathogenesis of EMC-D-induced diabetes using cyclosporin-A (CyA), a potent immunosuppressive drug. The data show that treatment with CyA results in increased severity and incidence of diabetes in susceptible ICR Swiss mice and induction of diabetes in resistant C3H/HeJ mice. It is concluded that immune mediation probably is not involved in the early pathogenesis of EMC-D-induced diabetes in mice.

A murine model of insulin-dependent diabetes mellitus resulting from the cumulative effects of the nondiabetogenic strain of encephalomyocarditis virus and a single low dose of streptozocin.

The induction of insulin-dependent diabetes in outbred male and female mice was examined using a combination of the usually nondiabetogenic B-variant of encephalomyocarditis (EMC-B) virus and single low doses of streptozocin (STZ). Neither EMC-B virus nor low doses of STZ were overtly diabetogenic when administered alone; however, when these two insults occurred 1 day apart, diabetes resulted in male but not in female mice. The induction of diabetes was dependent on the time interval between these two insults, since EMC-B virus and STZ given 4 days apart did not induce diabetes. Unexpectedly, when the order of these two insults was reversed, diabetes occurred. The absence of diabetes when EMC-B virus was given before STZ suggested the possibility that virus-induced interferon blocked the cytotoxic effects of STZ. This suggestion was supported by the observation that an antiserum against beta interferon abrogated the virus-mediated protection against STZ-mediated cytotoxicity. Also, Poly I:C administered before a single diabetogenic dose of STZ delayed the onset of severe hyperglycemia.

Exacerbation of the pathogenesis of the diabetogenic variant of encephalomyocarditis virus in mice by interferon.

Adult male ICR Swiss mice develop insulin-dependent diabetes when infected with the D variant of encephalomyocarditis virus (EMC-D). In contrast, adult C57Bl/6 males are relatively resistant to the diabetogenic effects of this virus. We have been studying the role of interferon (IFN) in the pathogenesis of infection by EMC-D and development of virus-induced murine diabetes mellitus. The data show that when IFN beta or IFN gamma were administered four days after virus infection, the frequency and severity of diabetes were exacerbated in ICR Swiss mice, and the diabetic state was induced in the resistant C57Bl/6 strain. In addition, animals treated with either of the IFNs or the IFN-inducer poly I:C, developed symptoms of severe encephalomyocarditis. Analysis of ICR Swiss mouse tissues revealed that IFN-treatment resulted in virus replication in the heart and brain and the reappearance of the virus in the pancreas. It is concluded that under certain conditions, the diabetic state is exacerbated and the normal course of (EMC-D)-infection in mice is altered by IFN.

Replication of diabetogenic and nondiabetogenic variants of encephalomyocarditis (EMC) virus in ICR Swiss mice.

The replication of the diabetogenic D (EMC-D) and nondiabetogenic B (EMC-B) variants of encephalomyocarditis virus in various tissues of the murine host was determined. Pancreatic insulin levels were also measured. EMC-D replicated in the spleen, pancreas, heart, lung, and intestines, while EMC-B was limited to the spleen and pancreas. EMC-B interfered with the replication of EMC-D in each of the tissues examined. Insulin levels were initially increased by both viruses. By 4 days postinfection, insulin levels were either normal or undetectable in (EMC-D)-infected animals, but were dramatically elevated in those infected with EMC-B.

Virus-induced diabetes mellitus in ICR Swiss mice is age dependent.

Adult male ICR Swiss mice develop diabetes mellitus when infected with the D variant of encephalomyocarditis virus. It was determined that the disease syndrome caused by the virus in this mouse strain is dependent upon the age of the animals. Mice younger than 7 weeks developed lethal encephalitis, whereas those past this age developed diabetes. The susceptibility of young mice to the diabetogenic effects of encephalomyocarditis virus was enhanced by testosterone.

Inhibition of virus-induced diabetes mellitus by interferon is influenced by the host strain.

The diabetogenic variant of encephalomyocarditis virus (EMC-D) induces a diabetes-like syndrome in certain strains of mice. A study was done to determine if virus-induced diabetes could be prevented by interferon (IFN). It was found that the production of diabetes by EMC-D was blocked by either IFN beta or a variety of IFN-inducers in SWR/J, but not ICR Swiss mice. The replication of EMC-D in cell culture was inhibited by IFN beta. It is concluded that the response of pancreatic beta cells to the protective effect of IFN, is probably under genetic control.

Effect of steroid hormones on virus-induced diabetes mellitus.

Female ICR Swiss mice, generally resistant to the diabetogenic effects of the D variant of encephalomyocarditis virus, develop diabetes to the same extent as males if they are pretreated with testosterone. The data suggest that testosterone is one of the factors involved in the development of diabetes in certain strains of mice.

Alteration of diabetes and interferon induction by the diabetogenic strain of encephalomyocarditis virus through cell passage.

The diabetogenic strain of encephalomyocarditis virus (D virus) was propagated in several continuous cell lines. Each virus stock was tested for its ability to produce diabetes in mice and induce L-cell interferon (IFN-beta). The effect of insulin on virus replication and IFN-beta induction was also determined. It was found that the severity of the diabetes and the amount of IFN-beta produced was dependent on the cell line used for virus propagation. Virus synthesis was augmented and IFN-beta production was altered in insulin-treated cell cultures. It is concluded that D virus either consists of more than one virus or that its diabetes and IFN-beta inducing characteristics are unstable.

Enhancement of viral RNA-induced interferon production of L cells treated with insulin and amphotericin B methyl ester.

A study was initiated to develop a more efficient method of producing high-titered L cell interferon. The induction of interferon by viral infectious RNA could be augmented by pretreating the cell cultures with insulin and infecting in the presence of Amphotericin B methyl ester (AmBME). L cells were treated for 24 hr with different concentrations of insulin and then challenged with MM virus RNA suspended in a balanced salt solution with and without AmBME. The fluids were collected 48 hr after infection and assayed for interferon activity. Interferon production was markedly increased in insulin-treated cultures and that the increase was dose-dependent.