Adenovirus myocarditis in mice

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

Adenovirus

Myocarditis

An Electron Z. R.

BLAILOCK,

(1968)

9,84-96

in Mice

Microscopic

E. R.

RABIN,

AND

Study’ J. L.

MELNICK

Baylor University Collegeof Medicine, Departments of Pathology and Virology, and Houston, Texas 77025

Epidemiology,

Received April

8, 1968

Adenoviruses are frequently encountered pathogens in man, especially in children. In a few reported instances of adenovirus pneumonia, electrocardiographic changes have suggested an associated myocarditis (Chaney e2 al., 1958; Drouhet, 1957). However, no human casesof adenovirus endocarditis have as yet been reported. The present report on murine adenovirus myocarditis in mice is one of a seriesof experimental studies on the pathogenesis of viral lesionsof the heart (Hassan et al., 1964; Rabin et al., 1964; Rabin et al., 1965). Characteristic adenovirus lesions of myocardium, mural endocardium, heart valves, and endothelium of the ascending aorta were observed by light and electron microscopy, or both. MATERIALS

AND

METHODS

A. Mice. Pregnant white Swiss mice (Webster strain and random bred) were obtained from Euer’s farm, Austin, Texas, and the Texas Inbred Mice Company, Houston, Texas. B. Tissue culture. Monolayers of mouse embryo cells were used for virus assay. MH lactalbumin hydrolyzate medium was used for growth and, following the development of a complete monolayer, ME maintenance medium was substituted. C. Virus. Suspensions of the mouse adenovirus were kindly supplied by Dr. W. P. Rowe (Hartley et al., 1960). D. Virus titrations were performed by plaque technique (Melnick, 1956), and the titers were expressed as the number of plaque forming units or PFU per milliliter of blood or as PFU per gram of tissue. E. Experimental design and light microscopy. Pregnant mice were kept under observation until litters were born, and then mice up to 7 days of age were selected. Mice u-ere inoculated intraperitoneally with a 0.1 ml suspensionof virus. Most Inocula were taken from a preparation which contained 0.3 X lo5 PFU per ml of virus. Animals were killed at intervals of up to 21 days after inoculation. Heart, kidney, and blood specimens were assayed for virus. Each specimen consisted of a pool from three animals. Heart and kidney specimens were frozen (-20°C) intact in sterile containers and stored until assayed. Blood specimens were collected aseptically from a throat incision and placed in sterile test tubes containing distilled water. * Presented March, 1967. This work

at

the

International

was supported

by NIH

Academy

of

Grants

HE-05435, 84

Pathology

Meeting,

HE-05584,

Washington,

CA-08803,

and

D.C.: AI-05382.

ADENOVIRUS

MYOCARDITIS

IN

MICE

85

Blood specimens were also stored at -20°C until assayed. For virus assay, tissue was ground in sterile porcelain mortars using alundum abrasive. A 10 % suspension of this material in isotonic saline was centrifuged and antibiotic agents were added to the clear supernatant fluid prior to assay. For light microscopy, tissues were fixed in 10% neutral buffered formalin or in 2.5 % glutaraldehyde in phosphate buffer. The formalin-fixed material was embedded in paraffin and stained with hematoxylin and eosin. The glutaraldehyde-fixed material was processed as described below for electron microscopy, and 1-p thick sections were stained with Paragon multiple stain. Examination of these sections provided the areas most suitable for study by electron microscopy. F. Electron microscopy. For electron microscopy, tissue was fixed in cold 2.5% glutaraldehyde in phosphate buffer pH 7.4 for 2 hours and then washed and stored in the phosphate buffer at 4°C until final processing (up to 1 week later). At that time, specimens were postfixed in 2% osmium tetroxide in cold Verona1 acetate pH 7.2 for 1 hour. Some specimens were fixed directly in 1% osmium tetroxide. All specimens were then dehydrated in increasing concentrations of ethyl alcohol and embedded in araldite. They were polymerized for 12 hours at 35”C, 8 hours at 45”C, and 12 hours at 60°C. Sections were cut on a Porter-Blum ultramicrotome and were placed on carbon-coated 200-mesh grids. They were stained with Reynolds lead citrate prior to viewing on an RCA EMU-SF microscope. RESULTS A. Virus assay. Virus was first detected in specimens of heart, kidney, and blood on postinoculation day 6 (Graph 1). At that time the titer was highest in the heart (2.3 X lo4 PFU/gm). The highest titers in the heart were on days 9 and 11 (greater than 4.0 X lo5 PFU/gm). The maximum virus titers in the blood (1.7 X lo4 PFU/ ml) and in the kidneys (greater than 4.0 X lo4 PFU/gm) were also reached on day 9. However, while the titer in the heart and blood fell rapidly after this time, it did not begin to fall in the kidneys until after day 15. No virus was recovered in the heart or blood specimens on days 13,15, or 17. B. Gross and light microscopeJindings. No grosschangeswere observed in autopsied animals until after day 7. Thereafter, scattered, off-white areas averaging 0.1 cm in diameter were noted in the myocardium and on epicardial and endocardial surfaces. Pleural and pericardial effusions were also seen after day 7. By light microscopy focal and confluent areas of necrosis and fragmentation of muscle fibers were seen beginning on day 6. Most of these lesions contained intranuclear inclusion bodies in the myocytes (Fig. lb). The cellular infiltrate at first (days 6-8) comprised small numbers of polymorphonuclear neutrophils. In later specimens(days 9-12) most of the infiltrates consistedof macrophages,lymphocytes, and plasma cells (Fig. lc). The smaller, early type lesions were also seen in the extensively damaged hearts from days 9-12 (Fig. la). Valve lesions contained cellular infiltrates similar in composition to those of the myocardial lesions as described above (Figs. 2a, 2b). Collagen bundles appeared fragmented and there was subendothelial edema. Intranuclear inclusions, similar to those noted in the myocardial lesions, were seenin endothelial cells and in fibro-

Z. R.

BLAILOCK,

E.

R.

RABIN,

AND

J.

L.

MELMCK

PROGESSIVE IIEALING AND CALCIUM DEPOSITION IN LESIONS OF THE FEW

Q

I

2

3

4

,

t

5

6

7

8

t

-

9

IO

II

12

i----------t ~ _.

---

13

14

.

.

I5

.-

I6

.

I7

DAYS The assay is summarized. animals.

of murine Each

adenovirus expressed point represents the

as PFU per gram of tissue concentration in a pooled

or milliliter specimen

of blood from three

of the lamina propria. Endothelial cells of the ventricular endocardium also contained intranuclear inclusion bodies. As the myocardial lesions progressed there was slight fibrosis, but the most striking feature of the healing lesions was marked dystrophic calcification in the necrotic areas (Fig. Id). This reaction was most prominent in the material from days 17 to 21. All the animals in the present series either died or were killed by day 21. Intranuclear inclusion bodies were noted in large numbers from day 6 to day 12; a few inclusions were still present at day 15. The inclusions were at first small, irregular and eosinophilic. In larger and more severe areas of damage, a variety of inclusions was seen (Fig. lb). Some were small and eosinophilic; others were large and exhibited varying degrees of basophilia. Most of the large basophilic inclusions were surrounded by a clear peripheral zone which separated them from marginated and condensed chromatin. Other organs, aside from the heart were damaged. Small lesions were noted in the tubules of the renal cortex and an occasional lesion was observed in the liver and in the adrenals. These lesions were seen between days 8 and 14. D. Electron microscopy. A wide variety of cells appeared infected. Developing adenovirions (70 mp) n-ere seen in the nuclei of myocytes, fibrocytes, and endothelial cytes

1. Photomicrographs of myocardium from mice infected with murine adenovirus. la. This lesion contains prominent intranuclear inclusion bodies in myocytes (arrows). There is also interstitial edema, necrosis of myofibers, and a mononuclear cellular infiltrate. Nine days postinoculation, H and E, x530 lb. Intranuclear inclusion bodies in myocytes are seen in detail. The smaller, lighter bodies appeared eosinophilic (E). The larger, darkly staining bodies are basophilic (B). Margination of chromatin and a clear zone around the iuclusions are also seen. Nine days postinoculation, H and E, Xl,600 :i lc. Myocardial necrosis is evident wit,h dense and prominent mononuclear cellular infil*.. trate. Nine days postinoculation, H and E, x450 ld. Uystrophic calcification is seen in a focus of myocardinl necrosis. Twelve days postinoculation, H and E, x550 FIG.

Q7

FIG. 2. Photomicrographs of heart valve from mice infected with murine adenovirus. 2~. Mitral valve leaflets are shown. A polymorphonuclear exudate covers the apposed endothelial surfaces. Both leaflets show necrosis, most marked in the leaflet on the right. H and E, X375 2b. Aortic valve leaflet is shown. Numerous intranuclear inclusion bodies (arrows) are present in fibrocytes. Phase contrast micrograph of 1-p thick section from araldite embedded tissue, Paragon Multiple Stain, X 1,750 88

ADENOVIRUS

MYOCARDITIS

IN

MICE

89

cells lining capillaries, ventricular endocardium, heart valves, and ascending aorta (Figs. 3 and 4). Virions appeared singly, in small aggregates, or in a crystalline arrangement in the nuclei of infected cells (Fig. lOa). Virus particles had central round cores (about 50 rnp) which varied in density (Fig. 10~). These cores were surrounded by a moderately dense, granular halo (5-15 rnp wide). Occasionally, virions had a coarsely granular surface. A variety of nuclear changes was present. Some nuclei were enlarged and contained a rounded mass of filamentous material which in some cases resembled nucleoli (Fig. 5). In some instances the margins of these dense masses were associated with small numbers of adenovirions (Fig. 7). Similar granular and filamentous material was seen in an irregular network-like arrangement in other infected nuclei (Fig. 8). These networks were usually associated with virus crystals and with rounded, granular, dense bodies 100-250 rnp in diameter (Fig. 9). Virus cytopathic effect was varied. In some cells, there was little or no apparent cytoplasmic reaction to the presence of large numbers of intranuclear virions. In infected myocytes there was frequently some dilatation of sarcotubular system and swelling of the mitochondria. Severe damage was evidenced by rupture of nuclei and spillage of virions and nuclear contents into the cytoplasm (Fig. 8). Disruption of the plasma membrane was also seen. In lesions of both myocardium and valves, nuclear and cytoplasmic debris was seen enclosed within cytoplasmic membranes of macrophages. Interstitial edema was particularly noticeable in the valve lesions (Fig. 3). Intracytoplasmic and extracellular virus was seen only in the immediate vicinity of cells undergoing lysis. DISCUSSION Murine adenovirus is not known to cause spontaneous disease in mice. The agent was initially recovered during attempts to isolate a mouse leukemia virus by Hartley and Rowe (1960). They found it produced a fatal systemic illness with myocarditis in inoculated suckling mice. Older mice and sucklings born of mothers with immunity to the virus were not susceptible. In our laboratory the virus in the dosage used produced a similar illness which was usually fatal in susceptible animals. Most of the animals died during the eighth to the twelfth postinoculation days, with only an occasional survivor to the turenty-first day. The infection was hematogenously disseminated, as virus was recovered by virus assay from blood, heart, and kidney. As in previous in vivo studies of experimental viral myocarditis carried out in our laboratories (Hassan et al., 1960; Rabin et al., 1965), the acute extensive myocardial injury coincided with highest virus titers in the heart, and with large numbers of virus particles often observed within disintegrating cells by electron microscopy. Consequently, the myocardial injury may be attributed to the cytopathic effect of direct viral invasion and multiplication. Among the various types of adenoviruses there is variation in the nuclear changes observed in infected cells (Bloch et al., 1960; Morgan et al., 1960; Morgan et al., 1959; Neubert, 1964; Sohier et al., 1965). The features consistently seenare: rounded virions 70 rnp in diameter; intranuclear growth; virion arrangement singly, in small aggregates, and in larger crystalline aggregates; and associated large eosinophilic and basophilic intranuclear inclusion bodies. All these features were observed in the

FIG. 3. Electron micrograph of aortic valve leaflet shows endothelial surfaces at top and lower right. Several small virus aggregates are present in the endothelial cell. Note the &all, very dense intranuclear bodies also present in this infected cell. The clear subendothelial space is interpreted as edema. X5,700. Insert shows virions from endothelial cell nucleus, X21,430 90

FIG. 4. Electron micrograph is from aorta near origin at aortic valve. Several virus crystalline arrays (arrows) are present in the upper endothelial cell. Beneath the endothelium there are elastic fibers and fibroblasts. X7,100 FIG. 5. Electron micrograph of myocyte nucleus shows dense body which may represent either an enlarged nucleolus or viral inclusion body. X12,000 FIG. 6. Electron micrograph of myocyte nucleus shows a large intranuclear viral inclusion which is slightly less dense than the structure described in Fig. 5.

91

FIG. 7. Electron micrograph of myocyte nucleus shows a large intranuclear inclusion with associated virions at its periphery. This inclusion body is less dense than those seen in Figs. 5 and 6. The nuclear envelope is intact. X 15,000 FIG. 8. Electron micrograph is of an infected myocyte nucleus. In this cell the inclusion material is in an irregular, network-like arrangement with virions at its margin and within its interstices (arrows). Note also the disruption of the nuclear envelope. X15,oCIo FIG. 9. Electron micrograph is of a myocyte nucleus showing in detail several adenovirions and a small intranuclear inclusion similar to those seen in nuclei with large virus crystals. Usually fine filaments radiated from these inclusions. Small rounded bodies (arrows) were also occasionally seen at the periphery. X85,700 03

FIG. 10. Electron micrographs show details of murine adenovirions in infected myocyte nuclei. 10a. Crystalline arrays of virions are seen displacing large areas of nucleoplasm. These are associated with small fragments of the inclusion material. X17,000 lob. Several virions at the margin of a fragment of inclusion material. Some virions (arrows) appear to be developing from the inclusion material. X90,000 10~. Detail of adenovirions in crystalline array. The core material of each virion is of variable density and is usually outlined by a ring about 50 mp wide. The virions are approximately 70-80 rnp in overall diameter. X55,000

94

Z.

R.

BLAILOCK,

E.

R.

RABIN,

AND

J.

L.

MELNICK

present study. Recently some of the intranuclear inclusions have been shown to contain structural protein of the virus (Stitch et al., 1967). The time sequence of viral replication is difficult to determine in the in vim system and is best done in vitro. However, in the present experimental model the observed nuclear changes suggestthe following as a possiblesequenceof viral replication: The infection seemedto proceed randomly from one cell to another, so that cells with various stages of viral development coexist. Penetration of the cell by virus leads to nuclear enlargement. A very dense inclusion material almost fills the nucleus, then becomeslessdense and is associated with small numbers of virions at its margin. Further viral replication occurs while the granulo-filamentous mass loosensinto a network-like structure with virus crystals at its margins and within its interstices (Fig. lOa). Some areas suggest that this granulo-filamentous material is intimately associatedwith production of adenovirions (Fig. lob). The role of the smaller dense bodies is unknown, but occasionally their margins were lined by 10 rnp granules (Fig. 9). The initial eosinophilic inclusion bodies may represent enlarged nucleoli. The larger granulo-filamentous massesassociatedwith virus replication appear to correspond to the large basophilic inclusions of more extensive lesionsin the H and E stained material and probably contain precursor viral structural protein. This sequenceof events is generally consistent with those described in tissue culture studies of other adenoviruses (Bloch et al., 1957; Morgan et al., 1960; Morgan et al., 1959). Nonviral protein crystals were not seen in the present study. It would seemthat many casesof viral infection of the heart in man go unrecognized. Such subclinical myocardial damage has been implicated as a possible cause for idiopathic myocardial hypertrophy, endocardial fibroelastosis, and diffuse myocardial fibrosis (Burch et al., 1967). The occurrence of viral endocarditis in man is uncertain, but chronic valvular diseaseappears in many patients having no history of rheumatic fever (Clawson et al., 1926). This fact led to the suspicion that other agents (Baggenstoss et al., 1960), including viruses (Burch et al., 1966), may cause endocarditis and subsequent valvular scarring in man. Previous investigators have described acute valvulitis in laboratory animals infected with coxsackievirus Bd (Burch et al., 1966) the encephalomyocarditis virus (Kilham et al., 1956), virus III (Pearce, 1960), and murine adenovirus (Blailock el al., 1967). These findings suggest that viruses may be a causeof someinstances of valvulitis in humans. Presumably, acute lesions, such aswe observed in the valves, may heal with resultant scar formation and dystrophic calcification, leading to valvular deformities which now are attributed to previous subclinical bacterial or rheumatic disease.Such a long term effect of viral infection has not yet been proven, although Burch (1967) has identified coxsackievirus antigen within damaged human heart valves and myocardium from routine autopsy specimens. The present report has shown that murine adenovirus not only can damage myocardium, but can also invade and destroy cells in cardiac valve tissue. Dystrophic calcification was a prominent feature in myocardial lesionsfrom the fifteenth to the twenty-first days. While such calcification was not observed in the valve lesions, our study of this phase of the infection has not been completed. It appears

ADENOVIRUS

MYOCARDITIS

that, such lesions could lead to focal myocardial valves in surviving animals.

IN

MICE

95

fibrosis and to scarred and calcified

SUMMARY Newborn white Swiss mice were inoculated intraperitoneally with suspensions of adenovirus. The dosages used produced a fatal illness characterized by an extensive myocarditis and a high incidence of acute valvulitis. Virus assay showed high titers of heart, blood, and kidney, corresponding to the time of most severe damage. Electron copy revealed intranuclear viral replication in myocytes, fibrocytes, and endothelial mural and valvular endocardium and of the aorta and myocardial capillaries. These may well have significance as animal model systems of myocardial and valvular lesions

murine acute virus in microscells of findings in man.

REFERENCES BAGGENSTOSS, 8. H., and SAPHIR, 0. (1960). Rheumatic diseases of the heart. “Pathology of the Heart,” 8. E. Gould, ed. 2nd ed., Thomas. BL.\ILOCK, Z. R., RABIN, E. R. and MELNICK, J. L. (1967). Adenovirus endocarditis in mice. Science 157, 69-70. BLOCH, D. P., MORGAN, C., GODMAN, G. C., HOWE, C., and ROSE, H. M. (1957). A correlated histochemical and electron microscopic study of intranuclear crystalline aggregates of adenovirus in HeLa cells. J. Riophys. Biochem. Cytol. 3, 1. BRANDOX, F. B., and MCLEAN, I. W. JR. (19G2). Adenoviruses.ddvances Virus Resecwch 9, 157. BURCH, G.E., DEPASQUSLE, N. P., SUN,S. C.,HALE, A. R., and MOGABGAB, W. J. (1966). Experimental coxsackievirus endocarditis. J. Am. iVIed. Assoc. 196, 349-352. BURCH, G. E., SUN, S. C., COLCOLOUGH, H. L., SOHSL, R. S., and DEPSSQU1LE, N. P. (1967). Coxsackie B viral myocarditis and valvulitis identified in routine autopsy specimens by immullofl~lorescellt techniques. Am. Heart J. 74, 13-23. CHANET, C., LEPINE, P., LELONG, M., LE-TAN-YINH, 8. P., and T'IRAT, J. (1958). Severe and fatal pneumonia in infants and younger children associated with adenovirus infection. :lm. J. Hyg. 67, 367-378. CLEMSON, B. J., BELL, E. T., and HARTZELL, T. B. (1926). Valvular diseases of the heart with special reference to the pathogenesis of old valvular defects. Am. J. Pathol. 2, 193-234. DROUHET, 1’. (1957). A fatal case of adenovirus pneumonia. Ann. Inst. Pasfew 93, 138-142. HARTLEY, J. W., and ROWE, W. P. (1960). A new mouse virus apparently related to the adenovirlls group. Virology 11, 645-637. H.issas, 8. A., R.IBIN, E. R., and MELNICB, J. L. (1964). Reovirus myocarditis in mice: an elect,ron microscopic, immunofluorescent and virus assay study. Expfl. Mol. Pafhol. 4, 66-80. KILHAY, L., MASON, P., and DAVIES, J. N. P. (1956). Host virus relation in encephalomyocarditis (E.M.C.) virus infections. II Myocardit,is in mongooses. Am. J. Trop. Med. Hyg. 5, 655. MELNICK, J. L. (1956). Tissue culture methods for the cultivation of poliomyelitis and other viruses. “Diagnostic Procedures for T?ral and Rickettsial Diseases,” Frances, T., ed. Amer. Public Health Assn. 97-152. New York. MORGAN, C., GODMAN, G. C., BREITENFELD, P. M., and ROSE, H. M. (1960). A correlative study by electron and light microscopy of the development of Type 5 adenovirus. J. Erpfl. dred. 112, 373-382. h’IoRo.IN, C., and ROSE, H. M. (1959). Electron microscopic observations on adenoviruses “Virus Growth and Variation,” Isaacs, A., and Lacey, and viruses of the influenza group. B. W., eds. Cambridge Univ. Press. NEUBEKT, G. (1964). Zur vermehrung des adenovirus typ 5. Zentralblatt fiir Bakferiologie, Parasifenkunde, Injection.skrankheiten, und Hygiene 194, 269-291. PE.IRCE, J. M. (1960). Heart disease and filterable viruses. Circulation 21, 448-455.

96

Z.

R.

BLAILOCK,

E.

R.

RABIN,

AND

J.

L.

MELNICK

E. R., HASSAN, S. A., JENSON, A. B., and MELNICK, J. L. (1964). Coxsackievirus B3 myocarditis in mice. Am. J. Path& 44, 775-797. RABIN, E. R., and MELNICK, J. L. (1965). Viral myocarditis. Cardiovascular Res. Bull. 4, 224. RABIN, E. R., PHILLIPS, C. A., JENSON, A. B., and MELNICK, J. L. (1965). VTaccinia virus myocarditis in mice: an electron microscopic and virus assay study. Exptl. and Mol. Pathol. 4, 98-111. SOHIER, R., CHARDONNET, Y., and PRUNIERAS, M. (1965). Adenoviruses, status of current knowledge. Prog. Med. Viral. 7, 253. STITCH, H. F., KALNINS, V. I., MACKINNON, E., and YOHN, D. S. (1967). Electron microscopic localization of adenovirus Type 12 antigens. J. Ultrastruct. Res. 19, 556-562. TEDESCHI, C. G., and STEVENSON, T. D. (1951). Interstitial myocarditis in children. NEJM 244, 352. RABIN,