VIRUS-INDUCED DIABETES MELLITUS I. HYPERGLYCEML~_ AND HYPOINSULINEMIA IN ~/IICE INFECTED WITH ENCEPHALOMYOCARDITIS VIRUS

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1 VIRUS-INDUCED DIABETES MELLITUS I. HYPERGLYCEML~_ AND HYPOINSULINEMIA IN ~/IICE INFECTED WITH ENCEPHALOMYOCARDITIS VIRUS BY D. WARK BOUCHER AND ABNER LOUIS NOTKINS (Frm the Virlgy Sectin, Labratry q[ Micrbilgy and Immunlgy, Natinal Institute f Dental Research, Natinal Institutes f Health, Bethesda, Maryland 214) (Received fr publicatin 15 December 1972) Fr nearly 75 yr it has been speculated that viruses might be ne f the causes f diabetes mellitus in man. In 1899, Harris reprted that diabetes develped in a patient shrtly after a mumps infectin (1). Since that time there have been numerus reprts shwing a tempral relatinship between the nset f viral infectins and the develpment f diabetes (2). A variety f viruses have been implicated in the etilgy f diabetes with mumps being the mst ppular candidate. Since, hwever, the incidence f bth diabetes and mumps is high, it is pssible that the relatinship between the tw might simply be frtuitus. Recently, it has been suggested that Cxsackie virus B4 als might prduce diabetes. Gamble et al. (3) fund that the titer f neutralizing antibdy t Cxsackie B4 was higher in newly diagnsed diabetic patients when cmpared with nndiabetic cntrls. The antibdy titer t a variety f ther viruses including mumps was nt significantly different frm cntrls. Althugh prvcative, a number f ther factrs might accunt fr these relatinships, and prf that viruses cause diabetes in man is still lacking. Evidence that viruses can prduce diabetes in animals als is sparse. In the early 196's a grup f investigatrs frm Italv bserved that cattle infected with ft-and-muth disease virus develped hyperglycemia and lesins in the pancreas (4, 5). Since nly a small grup f animals was studied these bservatins await cnfirmatin. Other viruses als can prduce lesins in the pancreas; but the islets f Langerhans ften were nt invlved r the animals were nt examined fr evidence f diabetes (6-9). Recently, Craighead and c-wrkers reprted that mice infected with the M variant f encephalmycarditis (EMC) t virus develped a diabeteslike syndrme (1, 11). Because f the ptential value f animal mdels fr understanding diabetes in humans, the present investigatin was initiated t study in greater detail the characteristics and duratin f the EMC-induced diabeteslike syndrme and t evaluate sme f the factrs respnsible fr its develpment. i Abbreviatins used in this paper: EMC, encephalmycarditis; IRI, immunreactive insulin; MEM, minimal essential medium THE JOURNAL OF EXPERIMENTAL MEDICINE VOLUME 137, 1973

2 D. WARK BOUCI-IER AND ABNER LOUIS NOTKINS 1227 Materials and Methds Cells.--Primary muse embry cultures were prepared by trypsinizatin f minced day ld CAF-1 embrys. Cells were grwn with Eagle's minimal essential medium (MEM) supplemented with 1% calf serum, 1 U f penicillin, 1 ~g f streptmycin, and 5 ~g f nemycin per ml. Animals.--DBA/2N and C57BL/6N mice were btained frm the breeding clny at the Natinal Institutes f Health. BALB/CJ, CBA/J, and A/J mice were purchased frm Jacksn Labratry, Bar Harbr, Maine. CD-1 mice were btained frm Charles River Breeding Labratries, Inc., Wilmingtn, Mass. Mice were maintained n Purina NIH Rat and Muse Ratin cntaining 5.% fat and 23.5% prtein. Animals were allwed free access t fd and water except where nted. Unless stated therwise, male mice were used in all experiments. Virus.--The M variant f EMC virus passaged in mice (MH22) was kindly supplied by Dr. J. Craighead (11, 12). 6-wk ld DBA/2N mice were infected intraperitneally (i.p.) with.1 ml f a 1:5 dilutin f this material. 5 days later a 1% muse heart suspensin was prepared in Eagle's MEM with 5% calf serum by hmgenizatin in a TenBreck all glass tissue grinder. A 1% suspensin f hearts frm uninfected mice was prepared in the same way. The virus titer was determined by inculating.2 ml f apprpriate dilutins f this material nt cnfluent mnlayers f primary muse embry cells in 15-mm plastic Petri dishes. After adsrptin fr 1 h at 37 C, cultures were verlayed with Eagle's MEM cntaining 5% calf serum and 2% methylcellulse (Methcel, 4 cps; Fisher Scientific C., Fairlawn, N. J.). Mnlayers were stained 4 days later with a 1 : 1, dilutin f neutral red and plaques were cunted within 12 h. The titer f the stck virus pl by this methd was 2.5 X 15 plaque-frming units (PFU)/ml. Fr titratin f virus in the pancreas a 1% suspensin was prepared by hmgenizatin as described abve. T avid pssible inactivatin f the virus by prtelytic enzymes released frm the pancreas, the hmgenate was titrated immediately fr infectivity. Analytical Methds.--Except where stated, 6-8-wk ld male DBA/2N mice were inculated i.p. with.1 ml f a 1:5 dilutin f nrmal muse heart suspensin r stck virus cntaining 5 PFU f EMC virus. Mrtality with this cncentratin f virus was 1-2%. Unless indicated therwise bld was btained frm nnfasted mice by bleeding them frm the retr-rbital plexus. Glucse was determined enzymatically by the glucse xidase methd (13) using -dianisidine dihydrchlride as the reactive dye (Sigma Chemical C., St. Luis, M.). Serum immunreactive insulin (IRI) was measured by the slid-phase radiimmunassay prcedure (14) with prcine insulin as the standard (Pharmacia, Uppsala, Sweden). Glucse and ketnes in the urine were measmed using Tes-tape and Ketstix btained respectively frm Eli Lilly and C., Indianaplis, Ind., and Ames C., Div. f Miles Lab., Inc., Elkhart, Ind. Glucse Tlerance Tests.--Mice were fasted fr 18 h and then inculated i.p. with a 23 aqueus slutin (wt/vl) f dextrse, 2 mg/g bdy weight. Animals were bled immediately befre injectin f dextrse ( time) and at 3, 9, and 12 min thereafter. The samples were then assayed fr bld glucse. RESULTS Hyperglycemia and Glycsuria in EMC-lrnfected Mice.--The first experiment was designed t study the time-curse and magnitude f the hyperglycemia induced by EMC virus. Male DBA/2N mice were infected with EMC virus, and at different times thereafter the animals were bled and bld glucse was determined. Mice inculated with a 1 % nrmal muse heart suspensin served as cntrls. Three separate experiments were perfrmed with apprximately

3 1228 VIRUS-INDUCED DIABETES MELLITUS 5 infected and 2 uninfected mice in each. The data in Fig. 1 represent the mean f these experiments. It can be seen that within 6-1 days after infectin the mean bld glucse level rse t greater than 3 rag/1 ml. Bld glucse then gradually declined but remained abve cntrls even at 17 days after infectin. Sme f these animals have nw been hyperglycemic fr ver 3 days. In cntrast, the mean bld glucse levels f mice inculated with a nrmal muse heart suspensin ranged frm 13 t 16 rag/1 m]. The mean glucse level f uninculated mice was fund t be 141 rag/1 ml with a stand E 3 ~ 26 W 1/ I (.9,-~ 18 _1 m 14 I I D AYS I I I I II FIG. 1. Mean bld glucse levels in DBA/2N male mice inculated with EMC virus r nrmal muse heart suspensin. At the times indicated animals were bled and bld glucse levels determined. Each pint represents the mean f 5-15 animals frm three separate experiments. In all experiments the vertical bars represent the standard errr f the mean (SEM). EMC virus, (e--o); nrmal muse heart, (O--O). ard deviatin (SD) f +29 and a standard errr f the mean (SEM) f 4-3. Less than 1% f these animals had bld glucse levels abve 2 rag/1 ml. In this paper, animals were cnsidered h33)erglycemic if their bld glucse levels exceeded 2 rag/1 ml (i.e., 2 SD greater than the mean f uninfected animals). A mre detailed analysis f the abve data, shwing the percentage f animals with different levels f glucse, is illustrated in Table I. 7 days after inculatin f the virus, 68% f the animals were h)~perglycemic while at 98 days nly 22 % were still hyperglycemic. N appreciable difference in mrtality was fund between the hyperglycemic and nnhyperglycemic grups.

4 D. WARK BOUCttER AND ABNER LOUIS NOTKINS 1229 TABLE I Percentage f Animals with Hyperglycemia After Injectin with EMC Virus* Grup Days pstinfectin Animals % Hyperglycemic (> 2 mg/1 ml) >4 mg/1 ml mg/1 ml Nnhyperglycemic (<199 rag/1 ml) * Data frm Fig. 1 expressed as the percentage f animals with different bld glucse levels. Data btained by fllwing bld glucse levels in individual animals revealed three general patterns. The mst cmmn pattern, fund in apprximately 6-8% f the animals, was a transient hyperglycemia lasting frm a few days t several mnths. The secnd pattern ccurring in 1-15 % f the animals was a persistent hyperglycemia lasting 6 r mre mnths. The third pattern fund in 15-3% f the animals was characterized by the failure t develp hyperglycemia. The data in Fig. 2 represent these patterns in three animals studied ver a 9 day perid. Infectin f mice with EMC virus als resulted in glycsuria. The data in Fig. 3 shw that in acutely infected mice there was a gd crrelatin between hyperglycemia and glycsuria. Animals with bld glucse levels belw 2 rag/1 ml had little r n sugar in the urine ( t 1+), while the majrity f animals with bld glucse levels abve 3 rag/1 ml had substantial amunts f sugar in the urine (3+ t 4+). Marked discrdance between glucse in the bld and urine was bserved, hwever, in abut 1% f the determinatins. Glycsuria als was detected in hyperglycemic animals infected fr 6 r mre mnths (data nt shwn). T see whether the hyperglycemia in EMC infectin was assciated with breakdwn f fat depsits, the urine f infected mice was tested fr ketnes. The data in Table II shw that althugh up t ne-third f the infected mice had lw levels f ketnes in their urine, there was n crrelatin between ketsis and hyperglycemia. Ketnes were nt detected in the urine f uninfected mice. Effect f Fasting n Bld Glucse Levels.--In the early phase f ur experiments we bserved that when animals were fasted vernight (18 h) the difference in bld glucse levels between infected and uninfected animals was bscured. T determine mre precisely the rate at which elevated bld glucse levels returned t nrmal, glucse determinatins were dne at different times after remval f fd. The data in Table III shw that within 12 h the mean bld glucse level f the infected grup apprached that f the uninfected

5 123 VIRUS-INDUCED DIABETES MELLITUS 5 r E 38 E 34 uj rn I I[- I I I I I I 3 5 "TO 9 DAYS l"tg. 2. Representative bld glucse patterns in mice after infectin with EMC virus Each curve represents data frm a single muse. Persistent hyperglycemic, (~--A); transient hyperglycemic, ( -- ) ; nnhyperglycemic, (O-- O ). grup. Only ne f the infected animals had a bld glucse level abve 2 rag/1 ml. As a result, nnfasted animals were used fr mst f ur studies. Fd and Water Cnsumptin in Infected Mice.--T study the effect f EMC virus infectin n fd and water cnsumptin, mice were divided int grups n the basis f their nnfasting bld glucse levels. Animals were placed in individual cages and the amunt f fd and water cnsumed by each muse ver a 6 wk perid was determined. The data in Table IV shw that infected nnhyperglycemic animals cnsumed apprximately the same amunt f water and slightly mre fd than uninfected animals. Hwever, infected hyperglycemic animals cnsumed almst twice as much water and significantly mre fd than the uninfected animals. There was n significant difference in weight gain amng the three grups. Impairment f Glucse Metablism in Infected Mice.--The data in Table I and Fig. 2 shwed that nt all animals infected with EMC virus became severely hyperglycemic. Mrever, in a large percentage f the animals that did becme hyperglycemic the bld glucse levels ultimately returned t nrmal. T see

6 D. WARK BOUCttER AND ABNER LOUIS NOTKINS ;"! (D 25 a g m 2 15 ".v 1 5 I I I I I GLYCOSURIA FIG. 3. Tempral relatinship between bld glucse and glycsuria. Fr each pint bld and urine specimens were btained within 4 h f each ther. Data were btained frm 86 mice that had been infected fr days. Each pint represents an individual animal. The line was cmputed by linear regressin analysis (crrelatin cefficient, r =.8). TABLE II Ketnes in the Urine f EMC-Infected Mice n. f mice with n. f mice with n. f kettic mice Time after infectin n. f mice tested bld glucse >2 with bld sugars mg/1 ml ketnes in urine * >2 rag/1 ml;t wk Cntrl * Ketne levels in the urine were determined with Ketstix strips and the amunt was recrded as small (l-f), mderate (2-F), r large (3+). In all but tw cases the amunt f ketnes in the urine did nt exceed 1-t-. :~ Bld and urine were btained within 4 h f each ther.

7 1232 VIRUS-INDUCED DIABETES MELLITUS TABLE III Bld Glucse Levels i~ Infected and Uninfected Mice after Remval f Fd Bld glucse (rag/1 ml) Grup n. f mice h after remval f fd Uninfected ± 151~ 144 ± 1 12 ± Infected* ± ± ± 9 * Animals had been infected fr 42 days. :~ Mean ± SEM. TABLE Fd and Water Cnsumptin in Infected and Uninfected Mice IV Bld glucse n. f (rag/1 ml) Water:~ Fd:~ cnsumptin cnsumptin Grup animals wk (ml/day) (mg/day) 6 Bdy weight (g) wk 6 Uninfected :E 8} 132 =k ::k == Infected* Nnhyper :J= ± ::=.2** glycemie Hypergly =E =E ]], [! 21.4 ::E.8 cemic ::k: * At start f the experiment ( wk) animals had been infected fr 28 days. :~ Fd and water cnsumptin was measured ver a 6 wk perid. Mean == SEM. I] P <.1 cmpared with uninfected animals. P <,1 cmpared with infected nnhyperglycemic animals. ** P <.5 cmpared with uninfected animals. whether there was residual impairment f glucse metablism in these animals, glucse tlerance tests were perfrmed. As expected, the hyperglycemic animals (bld glucse levels abve 2 rag/1 ml) displayed grssly abnrmal glucse tlerance curves (Fig. 4). Abnrmal glucse tlerance curves als were seen in infected animals that never had bld glucse levels greater than 2 Ing/1 ml (Fig. 4 A) and in infected hyperglycemic animals that nw had bld glucse levels less than 2 mg/1 ii11 (Fig. 4 B). The bld glucse levels in these animals were higher and remained elevated fr a lnger time than in uninfected animals. Thus, the ability f infected animals that were nt hyperglycemic t handle a glucse lad was impaired. Insulin Levels in Infected Mice.--T see whether the impairment f glucse metablism might be due t abnrmal insulin levels, infected animals were divided int hyperglycemic and nnhyperglycemic grups. The amunt f insulin in the plasma f fasted animals then was determined at different times after infectin. The data in Table V shw that the amunt f insulin in the plasma f the infected nnhyperglycemic mice was nt statistically different frm that f the uninfected mice. The insulin in the plasma f infected hyper-

8 D. WARK BOUCHER AND ABNER LOUIS NOTKINS A B 68 E 6 W 3 (,.,) E) J 9 O J m OO 12 [,,I l i 6,2 6,2 MINUTES FI. 4. Cmparisn f glucse tlerance tests in hyperglycemic and nnhyperglycemic mice. Mice were infected with EMC virus and divided int three grups: infected mice with bld glucse levels abve 2 mg/1 ml, (@--@); infected mice with bld glucse levels belw 2 mg/1 ml, (O--O); infected mice with bld glucse levels abve 2 rag/1 ml during the acute phase f the infectin, but belw 2 mg/1 ml at 1 wk after infectin, (O--v1). Uninfected mice with bld glucse levels belw 2 mg/1 ml (A--A) served as cntrls. Befre the glucse tlerance tests, the animals were fasted fr 18 h and their blnd glucse levels determined ( time). The mice then were injected i.p. with glucse (2 mg/g bdy weight) and bled at the times indicated. Each pint represents the mean f fur t eight animals. Animals had been infected fr 5 wk (A) r 1 wk (B). glycemic mice, hwever, was lwer than that f uninfected mice at each f the times tested, with highly significant differences at 56 and 133 days. The respnsiveness f EMC-induced hyperglycemia t exgenus insulin is illustrated in Table VI. It can be seen that within 3 min after injectin f insulin, there was a marked drp in bld glucse levels f bth the infected and uninfected mice. Over the next 9 min the bld glucse levels in bth grups began t rise; the bld glucse f uninfected animals returned t its preinsulin level, whereas the bld glucse in infected animals still was substantially belw its preinsulin level. Effect f Muse Strain and Sex n Bld Glucse Levels.--The data in Table

9 1234 VIRUS-INDUCED DL~BETES lviellitus TABLE V Immunreactive Insulin (IRI) in the Plasma f EMC-Infeeted Mice Grup IRI (~U/ml)* Days after infectin Uninfected :~ Infected Nnh)2~er NT glycemic Hyper- NT ,]1 glycemic NT = Nt tested. * Animals were fasted fr 18 h befre plasma was btained fr IRI determinatins. Each grup cntained frm 8-15 animals. :~ Mean 4- SEM. P <.1 cmpared with uninfected animals. ]1 P <.1 cmpared with infected nnh)~perglycemic animals. TABLE VI Changes in Bld Glucse after Injectin f Exgenus Ins~din* Grup n. f mice Bld glucse (rag/1 ml) min after insulin 3 6 i2 Uninfected 7 11 =t= 2~c Infected (hypergly cemic) * Animals were inculated i.p. with.6 U f prcine insulin. Bld fr the time determinatin was taken just befre injectin f the insulin. Mean ± SEM. VII and Fig. 5 shw that the develpment f hyperglycemia in EMC virus infectin was dependent upn bth the strain and sex f the animals. Of the six strains f male mice tested at 7 days after infectin, nly DBA/2N and CD-1 mice develped hyperglycemia (Table VII). Similar results were bserved at 14 days after infectin (data nt shwn). The lw bld glucse levels nted in A/J mice may be related t the mribund state f these animals. Cmparisn f bld glucse levels in EMC-infected DBA/2N male and female mice revealed the typical hyperglycemic respnse in males,, whereas the mean bld glucse levels in females did nt exceed 16 rag/1 ml (Fig. 5). In anther experiment with 8 infected female mice studied ver a perid f 35 days, nly 4 ut f 28 determinatins gave bld glucse values abve 2 mg/1 ml (data nt shwn). Tiler f Virus in the Pancreas.--T see whether the failure f female mice t develp hyperglycemia was related t the degree f viral replicatin, the amunt f infectius virus in the pancreas f male and female mice was determined.

10 D. WARK BOUCt~[ER AND ABNER LOUIS I'qOTKINS 1235 TABLE VII Bld Glucse Levels in Different Strains f Male Mice Infected with EMC Virus Strain Infected n. f Bld glucse* Percent Percent animals (mg/1 ml) hyperglycemic:~ mrtality* % % DBA/2N Jr ± CD ± ± CBA/J ± 7 Jr ± 2 3 C57BL/6N BALB/CJ ± 7 33 A/] q q * All mice were infected at 6 wk f age with 5 PFU f EMC virus. Bld glucse levels and percent mrtality were determined 7 days after infectin. :~ Animals were cnsidered hyperglycemic if their bld glucse levels were 2 SD greater than the mean f uninfected animals f the same strain. Mean :k SEM. 3O _ 26 E ~ 22 D _J 18 Q m ~ 14 I I I I I I DAYS FIG. 5. Bld glucse levels in DBA/2N male and female mice infected with EMC virus. Each pint represents the mean f 3-4 mice. Male, (@--@); female, (O--O).

11 1236 VIRUS-INDUCED DIABETES I~IELLITUS The data in Table VIII shw that there was little difference in virus titer between the tw grups. Infectius virus culd nt be recvered after 1 days even thugh hyperglycemia persisted in the males fr ver 6 m. TABLE VIII Titer f EMC Virus in the Pancreas f Male and Female Mice* Time after infectin Male Virus titer PFU (lgl~/g tissue) Female day :~ t <2. <2. 16 <2. NT 26 <2. NT 4 <2. NT NT = Nt tested. * Male and female mice were inculated i.p. with 5 PFU f EMC virus. At each f the times indicated six male and three female mice were sacrificed, and their pancreases were remved, hmgenized, and assayed fr infectius virus n primary muse embry cells. The data represent the mean f the individual determinatins. Mean -4-SD. DISCUSSION Craighead shwed that infectin f mice with EMC virus prduced a diabeteslike syndrme characterized by hyperglycemia and glycsuria (1). In the present paper we have cnfirmed and extended these bservatins. Our experiments shwed that the metablic abnrmalities were mst readily detected by measuring the bld glucse levels f nnfasted animals and by perfrming glucse tlerance tests n fasted animals. This was necessitated by the bservatin that the bld glucse levels f the infected hyperglycemic mice remained elevated fr nly a shrt time after remval f fd, making it difficult t detect metablically abnrmal animals. Measurement f glucse in the urine was a useful screening prcedure fr detecting hyperglycemic animals, but it did nt prvide the fine quantitatin necessary t distinguish animals n the basis f the severity f their disease. Analysis f the bld glucse data frm hundreds f EMC-infected animals revealed that the metablic abnrmalities fit a spectrum ranging frm very mild alteratins detected by glucse tlerance tests t very severe alteratins detected by simply measuring the bld glucse levels f nnfasted animals. By dividing animals int grups n the basis f the severity f their hyperglycemia, it was pssible t detect metablic abnrmalities that therwise might have been bscured r cmpletely missed. In additin t hyperglycemia and

12 D. WARK BOUCHER AND ABNER LOUIS NOTKINS 1237 glycsuria, the severe frm f the diabeteslike syndrme is characterized by plydipsia, plyphagia, and hypinsulinemia. Lw levels f ketnes als were fund in the urine, but whether ketsis is part f the syndrme r a sequela f the mre generalized EMC infectin is nt clear. By cmparing severely hyperglycemic with nnhyperglycemic animals, it shuld be pssible t mre accurately assess the varius metablic abnrmalities and determine whether the vascular changes ften assciated with human diabetes (e.g. glmerulsclersis and retinpathy) ccur in the animal mdel. Several lines f evidence suggest that the metablic abnrmalities f the diabeteslike syndrme are due t pancreatic damage and hypinsulinemia. First, infectius virus can be recvered frm the pancreas during the acute phase f the infectin. Secnd, the infectin results in diminutin in the number and size f the islets f Langerhans and marked degranulatin f beta cells (1, 11, 15). Third, decreased levels f IRI in the pancreas f infected mice have been reprted, but the effect f EMC infectin n the IRI levels in the bld has been difficult t evaluate because f wide variatin in the data and small numbers f animals (11). By using large numbers f animals and dividing them int severely hyperglycemic and nnhyperglycemic grups, we have been able t shw that levels f IRI were significantly reduced in the bld f the hyperglycemic animals. The bservatin that the level f IRI was nt as greatly depressed in acutely infected hyperglycemic animals as cmpared with lngterm hyperglycemic animals (Table V) nce again may simply reflect a difference in the ppulatin f animals sampled (i.e., we d nt knw what percentage f animals tested n days 8, 14, and 28 belnged t the transient as cmpared with the mre severe lng-term hyperglycemic categry). Finally, the demnstratin that the bld glucse levels f the hyperglycemic animals culd be lwered by injecting exgenus insulin supprts the cntentin that the EMC-induced diabeteslike syndrme is, at least in part, secndary t insulin deficiency. Why EMC virus prduces severe and prlnged hyperglycemia in sme animals, transient hyperglycemia in thers, and mild if any alteratins in glucse metablism in still thers is nt knwn. Althugh the marked difference in incidence f diabetes between strains might be explained n a genetic basis, it is harder t explain the different respnses bserved in the DBA/2N males since these are inbred animals. One pssibility that is being investigated is that subtle differences in the time f initiatin and magnitude f the immune respnse may determine whether r nt the virus prduces severe r mild damage t the pancreas. It des nt appear that the lw incidence f hyperglycemia in female as cmpared with male DBA/2N mice can be explained simply n the degree f viral replicatin, since apprximately the same amunt f infectius virus was recvered frm the pancreas in bth sexes. Mrever, preliminary experiments have failed t reveal any significant differences in the titer f virus in the pancreas f infected hyperglycemic as crn-

13 1238 VIRUS-INDUCED DIABETES MELLITUS pared with nnhyperglycemic males at 4, 6, and 8 days after infectin. The titer f virus in the hmgenate f the whle pancreas, hwever, may nt reflect differences in the degree f viral replicatin in the beta cells f these animals. Alternately, hrmnal differences may make the pancreas in the male mre susceptible t virus-induced injury. In this cnnectin, it is knwn that EMC virus prduces mre severe disease and higher mrtality in males than females (16). The bservatin that hyperglycemia persists in the males fr many mnths after infectius virus can be recvered frm the pancreas suggests that the infectin prduces acute beta cell damage that in sme cases results in permanent impairment f functin. The pssibility des exist, hwever, that the virus may persist in the pancreas f these animals at lw levels r in a latent frm. The demnstratin that EMC virus can prduce in labratry animals a picture that metablically resembles diabetes adds credence t the hypthesis that viral infectins might be a cause f sme frms f diabetes in man (2, 17). Viruses that subtly r grssly damage the pancreas f animals r man wuld be gd candidates fr further study. It is evident frm ur experiments, hwever, that the develpment f diabetes is nt nly dependent n the nature f the virus, but n the cmplex interactins between the virus and the hst. SUMMARY Infectin f DBA/2N male mice with encephalmycarditis virus resulted in a diabeteslike syndrme characterized by hyperglycemia, glycsuria, hypinsulinemia, plydipsia, and plyphagia. Bld glucse levels were elevated within 4 days after infectin and reached a maximum mean level f 32 mg/ 1 ml within 12 days. Apprximately 6-8% f the animals develped a transient hyperglycemia while 1-15 % f the animals remained hyperglycemic fr well ver 6 t. The remaining animals failed t becme hyperglycemic but many had abnrmal glucse tlerance curves. Hyperglycemia was mst prnunced when animals were allwed free access t fd, and the incidence f hyperglycemia was related bth t the strain and sex f the animals, with few females develping hyperglycemia. The amunt f immunreactive insulin in the plasma f infected hyperglycemic mice was significantly lwer than in apprpriate cntrls, and injectin f exgenus insulin resulted in a rapid drp in the bld glucse levels. Despite the fact that certain animals were hyperglycemic fr many mnths, virus culd nt be recvered frm the pancreas after the first 1 days f the infectin. The authrs gratefully acknwledge helpful discussins with Dr. Jhn Craighead and the technical assistance f Ms. Teclia Brwn. REFERENCES 1. Harris, H. F A case f diabetes mellitus quickly fllwing mumps. Bstn Med. Surg. J. 14: Levy, N. L., and A. L. Ntkins Viral infectins and diseases f the endcrine system. J. fnfect. Dis. 19.4:94.

14 D. WARK BOUCttER AND ABNER LOUIS NOTKINS Gamble, D. R., M. L. Kinsley, M. G. Fitzgerald, R. Bltn, and K. W. Taylr Viral antibdies in diabetes meuitus. Br. Med. J. 3: Barbni, E., and I. Mancchi Alterazinia pancreatic in bvini cn diabete mellit pst-afts. Arch. Vet. Ital. 13: Barbni, E., I. Mancchi, and G. Asdrubali Observatins n diabetes mellitus assciated with experimental ft-and-muth disease in cattle. Vet. Ital. 17: Pappenheimer, A. M., L. J. Kunz, and S. Richardsn Passage f Cxsackie virus (Cnnecticut-5 strain) in adult mice with prductin f pancreatic disease. J. Exp. Med. 94: Dalldrf, G., and R. Giffrd Adaptatin f Grup B Cxsackie virus t adult muse pancreas. J. Exp. Med. 96: Burch, G. E., C. Tsui, J. M. Harb, and H. L. Clclugh Pathlgic findings in the pancreas f mice infected with cxsackievirus B4. Arch. Intern. Med. 19.8:4. 9. Tsui, C. Y., G. E. Burch, and J. M. Harb Pancreatitis in mice infected with cxsackievirus B 1. Arch. Pathl. 93: Craighead, J. E., and M. F. McLane Diabetes mellitus: inductin in mice by encephalmycarditis virus. Science (Wash. D. C.). 162: Craighead, J. E., and J. Steinke Diabetes mellitus-like syndrme in mice infected with encephalmycarditis virus. Am. J. Pathl. 63: Craighead, J. E Pathgenicity f the M and E variants f the encephalmycarditis (EMC) virus. I. Mycarditrpic and neurtrpic prperties. Am. J. Pathl. 48: Raab, E., and T. C. Terkildsen On the enzymatic determinatin f bld glucse. Scand. J. Clin. Lab. Invest. 19.: Wide, L., and J. Prath Radiimmunassay f prteins with the use f Sephadex-cupled antibdies. Bichim. Biphys. Acta. 13: Mfintefering, H., W. A. K. Schmidt, and W. KSrb Zur Virusgenese des diabetes mellitus bei der weiben maus. Dtsch. Med. Wchenschr. 96: Friedman, S. B., L. J. Grta, and L. A. Glasgw Differential susceptibility f male and female mice t encephalmycarditis virus. Effects f castratin, adrenalectmy, and the administratin f sex hrmnes. Infect. Immun. 5: Editrial Cxsackie viruses and diabetes. Lancet. 2:84.