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.

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.

Role of interferon in the propagation of MM virus in L cells.

MM virus propagated in mouse brain replicates to low titers in L cells without production of cytopathic effect (CPE). After growing the virus in BHK-21 cells, however, the virus replicates to high titers in L cells with complete CPE. It was found that suspensions of MM virus propagated in L cells directly from the mouse brain contained much more interferon than did suspensions of virus which had first been grown in BHK-21 cells. Mouse brain suspensions of the virus were also found to contain high interferon titers. Treatment of L cells with actinomycin D before infection with mouse brain-grown virus resulted in full virus replication with CPE. BHK-21 cell-grown virus diluted in L cell interferon behaved like mouse brain-grown virus in L cells. It is concluded that the presence of interferon in the inoculum is largely responsible for the suppression of MM virus replication in L cells.

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.

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.

Effect of Estrogen and Other Steroids on MM Virus Infection in Mice.

The effect of several steroid hormones on the susceptibility of mice to infection with MM virus was studied. Estrone, cortisone, and hydrocortisone increased mortality, whereas progesterone, prednisolone, and testosterone had no effect. The viral infection-enhancing (VIE) activity of estrone was maximal when the hormone was given 24 to 72 hr prior to viral inoculation. Less VIE activity was seen when the estrone was administered at the same time as the virus, and hormone treatment 24 hr after inoculation had no significant effect on mortality. Virus was found in the blood 24 hr before it appeared in the brain, regardless of estrone treatment. However, viremia was demonstrated in estrone-treated mice 48 hr before it occurred in control animals. There was no significant difference in the respective titers reached in the sera or the brains of the two groups.

Rapid sensitive assay for interferons based on the inhibition of MM virus nucleic acid synthesis.

A method for assaying mouse interferon based on the inhibition of viral ribonucleic acid (RNA) synthesis was devised. The amount of MM virus and RNA synthesized in interferon-treated L-cell cultures was determined by measuring the amount of (3)H-uridine converted into a trichloroacetic acid-insoluble form after treatment of the infected cultures with 2.5 mug of actinomycin D per ml. The amount of RNA synthesized was inversely related to the concentration of interferon used for treatment. A linear dose-response regression curve was obtained by plotting the log of the amount of RNA made, expressed as a percentage of the control, versus the log of the reciprocal of the interferon dilution. A unit of interferon was defined as that concentration which inhibited nucleic acid synthesis by 50% (INAS(50)). The concentration of mouse interferon could be determined within 24 hr. This assay method, on the average, was approximately half as sensitive as the method which measured the 50% reduction of MM virus plaque number (PDD(50)-MM method), but was, on the average, almost 1.7 times as sensitive as the PDD(50)-VSV method. It averaged approximately 20 times the sensitivity of the methods which used as end points the 70% reduction in yield of MM virus or the complete inhibition of cytopathic effect by MM virus. The reproducibility of the INAS(50) technique was tested in two ways. (i) Four independent assays of an interferon specimen were performed with replicate cultures. The standard deviation was 11.2% of the mean titer. (ii) On different dates, one interferon specimen was assayed seven times and another was assayed four times. The standard deviations were 21.5 and 26.6% of the respective mean titers.

Propagation of MM virus in L cells.

MM virus (mouse-brain stock) replicated to a limited extent in L cells without cytopathic effects; the average yield was less than 1 plaque-forming unit/cell. Passage in BHK-21 cells resulted in MM virus [MM(BHK)] which replicated to high titers (200 to 300 plaque-forming units/cell) in L cells with complete cytopathic effects. Appearance of mature MM(BHK) virus in L-cell cultures begins 4 hr after infection and is completed by 8 hr. Release of mature virus was slow (less than 1% at 8 hr) but was completed by 24 hr.

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.

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.

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.

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.

Effect of progesterone and testosterone on interferon production and on the viral infection-enhancing activity of estrone and hydrocortisone.

The effect of progesterone and testosterone on interferon production and on the viral infection-enhancing (VIE) activity of estrone and hydrocortisone was investigated. Neither hormone interfered with interferon production. Progesterone reduced the VIE activity of estrone but not that of hydrocortisone. Testosterone had no effect on the VIE activity of either hormone. It is concluded that the lack of VIE activity by progesterone and testosterone may be related to their inabilities to suppress interferon production. The interference by progesterone with the VIE activity of estrone suggests that, in this system, the two hormones may affect common target cells.

Tilorone hydrochloride: lack of correlation between interferon induction and viral protection.

The protection of mice against MM virus infection and the induction of circulating interferon by tilorone hydrochloride were determined. Whereas protection was evident with doses of 0.15 and 1.5 mg/kg, interferon was not detected with doses lower than 150 mg/kg. Protection was apparently not dependent on interferon induction.

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.

Identification of an interferon in murine placentas.

It is becoming increasingly apparent that interferon exerts a major role in cellular regulatory processes. Besides its well known antiviral properties, interferon has been shown to affect cell surface antigen expression, lymphoid cell function, as well as cellular protein synthesis and growth. To date, however, there is little information available on the mechanism of interferon production in vivo except in response to a variety of exogenously administered viral and nonviral systemic inducers. We report here the detection of a pregnancy-associated elevation of interferon, or an interferon-like component, in selected reproductive tissues of mice.