Jun 24, 1975 - The M variant of encephalomyocarditis virus is very closely related serologi- cally to five other strains of encephalomyocarditis virus. Despite ...
Vol. 12, No. 5 Printed in USA.
INFECTION AND IMMUNITY, Nov. 1975, p. 1224-1226 Copyright (C 1975 American Society for Microbiology
Virus-Induced Diabetes Mellitus V. Biological Differences Between the M Variant and Other Strains of Encephalomyocarditis Virus MICHAEL E. ROSS, TAKASHI ONODERA, KOZABURO HAYASHI, AND ABNER L. NOTKINS* Laboratory of Oral Medicine, National Institute of Dental Research, Bethesda, Maryland 20014
Received for publication 24 June 1975
The M variant of encephalomyocarditis virus is very closely related serologically to five other strains of encephalomyocarditis virus. Despite the serological relationship, these five viruses differ markedly from the M variant in their tissue tropisms and only the M variant infects beta cells of the pancreas, producing diabetes.
Encephalomyocarditis virus (EMC) has been recovered from a variety of animal sources. Although somie of these isolates have been named separately, they have been difficult to differentiate serologically (3, 5, 7). The M variant of EMC virus is unusually interesting because of its ability to infect pancreatic beta cells in mice and to cause a syndrome similar to human juvenile diabetes mellitus (2, 4). The E variant of EMC, which was isolated from the same source as the M variant, also may cause diabetes (8), but other strains of EMC have not been examined for their ability to damage the islets of Langerhans and produce diabetes. The present study was initiated to compare the serological properties and biological behavior of the M variant with other strains of EMC. The M variant of EMC was obtained from J. Craighead; stock virus was prepared from mouse hearts 4 days after infection. Prototype EMC was purchased from the American Type Culture Collection, Rockville, Md.; Mengo, Kissling, Columbia SK, and Columbia MM viruses were gifts from R. Shope and J. Casals of Yale University. With all of these viruses stock pools were prepared from the brains of newborn mice 24 h after intracerebral infection. Viruses were titrated on secondary mouse embryo cells, using a plaque assay with methylcellulose overlay. Adult male NIH mice, obtained from our mouse colony, were allowed free access to food and water throughout the study. Blood glucose levels were measured using a glucose oxidase assay on blood obtained from the retro-orbital venous plexus (2). For glucose tolerance tests, the blood glucose level was measured 60 min after an intraperitoneal injection of 2 mg of glucose per g of body weight. Glucose assays and glucose tolerance tests were performed 7 to
14 days after infection. For the M variant, 500 plaque-forming units (PFU) of virus was used as the infecting inoculum. For determination of the mean lethal dose of the other EMC viruses, serial 10-fold dilutions of virus were used. In all cases animals were infected by the intraperitoneal route. Antibody to the M variant of EMC was harvested from mice infected 3 weeks earlier with 500 PFU of virus; antibody to Mengo virus was harvested from guinea pigs 3 weeks after a single intraperitoneal infection with 1,000 PFU of virus. Neutralization titers were determined by incubation of 400 PFU of virus in a volume of 0.2 ml with an equal volume of serial twofold dilutions of serum for 1 h at 37 C. Fifty percent reduction in the number of plaques served as the end point. Antibody to the M variant of EMC was used for all immunofluorescence studies (6). Immunofluorescence was performed either by the direct technique, with anti-EMC antibody conjugated with fluorescein isothiocyanate, or by the indirect technique, with fluorescein isothiocyanatelabeled rabbit anti-gamma globulin. Pancreas, heart, and brain sections, obtained 3 to 7 days after infection, were stained with either fluorescein-labeled antibody or hematoxylin and eosin and examined microscopically. The serological relationship between the M variant of EMC virus and the other strains of EMC is illustrated in Table 1. Antibody to the M variant of EMC neutralized the M variant and all the other strains to approximately the same degree. Antibody to Mengo virus neutralized both Mengo and the M variant of EMC. Because the M variant of EMC behaved serologically like the other strains, we compared the ability of these viruses to produce diabetes. The M variant of EMC caused an essentially
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nonlethal infection in adult mice infected with the islets of Langerhans of mice infected with 500 PFU of virus. However infection produced the five other strains of EMC virus. However, hyperglycemia or abnormal glucose tolerance mice infected with these strains showed modertests in about 70% of the mice. In contrast, all ate to large amounts of viral antigen in the of the other strains of EMC killed the vast brain and meninges and variable amounts of majority of the animals, even with as little as antigen in the myocardium and the acinar pan10 PFU of virus. The deaths occurred between 3 creas. Again, the immunofluorescence data corand 10 days after infection. None of the mice related well with the changes seen by routine surviving the infection manifested either hy- histology. perglycemia or an abnormal glucose tolerance A close antigenic relationship between prototest. Moreover, mice dying of infection showed type EMC, Mengo, Kissling, Columbia SK, and no evidence of hyperglycemia when it was Columbia MM viruses previously has been repossible to measure glucose levels before death. ported (3, 5, 7). The demonstration here of neuBecause high mortality made it difficult to tralization of these five viruses by antibody to measure glucose levels, damage to pancreatic the M variant of EMC and neutralization of the islets also was assessed by immunofluorescence M variant by antibody to Mengo virus shows and histopathology (Table 2). Mice infected that the M variant is antigenically closely rewith the M variant of EMC showed large lated to the other strains of EMC virus. Howamounts of viral antigen in the pancreatic is- ever, these findings do not exclude the possibillets and lesser amounts of viral antigen in the ity that there are unique antigenic determimyocardium. These immunofluorescence find- nants on the M variant of EMC or other viruses ings correlated well with the amount of tissue in this group which were not detected by simple damage seen in sections obtained with hematox- neutralization. Despite the close serological reylin and eosin. No viral antigen was detected in lationship, the biological behavior of these viruses is very different. The prototype EMC and the four other strains tested all produced a TABLE 1. Serological relationship between the M lethal infection characterized by encephalitis or variant ofEMC virus and other strains ofEMC virus meningoencephalitis. Damage to the myocardium and acinar pancreas was mild, if present Neutralization titera at all. The islets of Langerhans were spared Virus Antibody to Antibody to completely. In contrast, the M variant did not M variant of Meng nervous system disease in adult mice, cause 0 nrus EMC virus and produced only mild myocarditis. However, the M variant infected the islets of Langerhans, M variant ......... 1,600 1,600 NDb 1,600 Prototype ......... causing moderate to severe beta cell necrosis 1,600 Mengo ...... ... 1,600 and diabetes. ND 800 Columbia SK ......... Many factors, including genetic differences ND Columbia MM ......... 800 among strains of mice (1, 2), are known to affect ND Kissling ......... 1,600 the development of diabetes after infection with the M variant of EMC. In the present a dilution serum producReciprocal of the highest we have used only male NIH mice; study, ing a 50% reduction in viral plaques. b
whether the beta cells of other strains of mice
ND, Not done.
TABLE 2. Detection of viral antigens by immunofluorescence after infection of mice with different strains of EMC virusa Virus
Mean lethal
Islets of
(doeFU)
Langerhans
M variant .................. >500 10 Prototype .................. Mengo ..................... 10 10 Kissling ................... Columbia SK .............. 10 Columbia MM .............. 10
Acinar pancreas
Heart
++
0
0 0 0 0 0
+
+ +
Nervous system
0
0
0
+
+
+++ ++
0 0
0 0
++ ++
a Sections were prepared from at least three animals that had been infected 3 to 7 days earlier and stained with fluorescein-labeled antibody. Symbols: 0, No viral antigen; +, small amounts of antigen, focal distribution; + +, moderate to large amounts of viral antigen, widely distributed.
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might be more susceptible to different strains of EMC virus is not known. The present study does show that differences between closely related viral strains are as important as genetically determined host differences in producing diabetes. LITERATURE CITED 1. Boucher, D. W., K. Hayashi, J. Rosenthal, and A. L. Notkins. 1975. Virus-induced diabetes mellitus. III. Influence of the sex and strain of the host. J. Infect. Dis. 131:462-466. 2. Boucher, D. W., and A. L. Notkins. 1973. Virus-induced diabetes mellitus. I. Hyperglycemia and hypoinsulinemia in mice infected with encephalomyocarditis virus. J. Exp. Med. 137:1226-1239. 3. Craighead, J. E. 1965. Some properties of the encephal-
4.
5. 6. 7.
8.
omyocarditis, Columbia SK and Mengo viruses. Proc. Soc. Exp. Biol. Med. 119:408-412. Craighead, J. E., and J. Steinke. 1971. Diabetes mellitus-like syndrome in mice infected with encephalomyocarditis virus. Am. J. Pathol. 63:119-134. Dick, G. W. A. 1949. The relationship of Mengo Encephalomyelitis, Encephalomyocarditis, Columbia-SK and M.M. viruses. J. Immunol. 62:375-386. Kawamura, A., Jr. (ed.). 1969. Fluorescent antibody techniques and their applications. University Park Press, Baltimore. Warren, J., J. E. Smadel, and S. B. Russ. 1949. The family relationship of encephalomyocarditis, Columia-SK, M.M., and Mengo Encephalomyocarditis Viruses. J. Immunol. 62:387-398. Wellmann, K. F., B. W. Volk, P. Brancato, and D. Amsterdam. 1973. Encephalomyocarditis virus (E variant) in mice. Pancreatic changes. Arch. Pathol. 95:304-311.
