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Ultrastructural cytopathology of mouse myocardium associated with EMC viral infection
~oburnal of Molecular
and Cellular Cardiology
(1973)
5,55-62
Ultrastructural Cytopathology Associated with EMC
Department
of Medicine
of Mouse Myocardium Viral Infection*
J. M. HARB AND G. E. BURGH of the Tulane University School of Medicine Louisiana, (Received
22 May
.New Orleans,
Louisiana,
and th
Charity
Hospital
of
U.S.A.
1972, and accepted 24 August
1972)
J. M. HARB AND G. E. BURGH Ultrastructural Cytopathology of Mouse Myocardium Associated with EMC Viral Infection. Journal of Molecular and Cellular Cardiology (1973) 5, 5562. Lesions in the mouse myocardium are described which are fairly characteristic of cytopathology caused by direct infection with EMC virus. The lesions consisted of formation of numerous cytoplasmic membrane-bound vesicles and vacuoles. Viral crystals were frequently associated with these lesions in the newborn mouse. Accompanying the lesion was a breakdown of the myofibrils, mitochondria, sarcoplasmic reticulum and transverse tubular system. The absence of these organelks clearly indicates a reduced contractability of the damaged myocytes. Possible factors resulting in membrane proliferation and mechanisms for cell lysis are discussed.
1. Introduction We have described experimental infections of mice with encephalomyocarditis (EMC) virus, Coxsackie viruses B1 and B4, ECHO 9 virus and mycoplasma gallisepticum agent [453. These studies have included investigations of the damage caused by viral infection to the myocardium [ 22, 471, endocardium [8], valves [II, 19, 231, aorta [14], coronary arteries [14], pancreas [15, 16, 20, 481, liver [17] and kidneys [ 281. It has been shown that myocarditis can be produced by many viruses, including reovirus [29], vaccinia virus [38], adenovirus [S], and Coxsackie virus Bs [37j, as well as those mentioned above. Viral damage to the heart may also appear as mural endocarditis [4, 9, 10, 19, 421, valvulitis [S, II, 13, 23, 431, pericarditis [46], or pancarditis [7, 441. The EMC virus, as used in this study, has been shown to cause myocardial damage in a number of animals [IZ, 24, 25, 28, 31, 33, 34, 35, 36, 411. The EMC virus was initially recovered from anthropoid apes [30] and was later isolated also from Aotus monkeys [40]. In our studies of EMC virus infections of the myocardium of the mouse [12], we noted a peculiar and somewhat characteristic ultrastructural cytopathology * Supported by grant HE-06769 from the National Heart and Lung Institute of the U.S. Health Service, the Rudolph Matas Memorial Fund for the Kate Prewitt Hess Laboratory, Rowe11 A. Billups Fund for Research in Heart Disease and the Feazel Laboratory
Public the
56
J. hf. HARB
AND
G. E. BURGH
associated with the formation of viral crystals. It is the purpose of this paper to report and to describe this cytopathology in detail.
2. Methods
and
Materials
A total of 39 newborn and 15 S-day-old Swissmice were inoculated with 0.05 ml or 0.1 ml of EMC virus culture fluid with a titre of 10-e TCID~(I. Thirteen control Swissmice (nine newborn and four 8-day-old animals) were inoculated with 0.1 ml of virus-free culture fluid. The mice were killed 24 h after inoculation, except for one control mouse which was killed 2 days after inoculation. Five of the infected newborn mice were found dead in their cages 24 h after inoculation. These five mice were included in this study along with the others. A portion of the left ventricle was excised immediately after the mice were killed by cervical spinal dislocation. The tissueswere minced into 1 mm3 pieces in a pool of cold 3% phosphate-buffered glutaraldehyde. The piecesof tissuewere then placed in fresh glutaraldehyde for 2 h. Following a 3-h rinse with 6 to 8 changes of fresh phosphate buffer, the tissueswere post-fixed for 13 h in cold 1o/ophosphatebuffered osmium tetroxide. Dehydration was accomplished in a graded series of alcohol. After infiltration with propylene oxide, the tissueswere placed in a mixture of 3 propylene oxide and JJepon and left under a lamp overnight. The tissues were then put through two l-h changes of pure epon and were finally embedded in preformed capsules. The tissueswere allowed to cure in an oven at 60°C for 72 h. Thin sections (400 to 600 A) were cut using both glass and diamond knives. The sectionswere stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop I electron microscope at initial magnifications of 10 000, 14 000 and 18 000 diameters. Viral crystals were readily visible at a magnification of 14 000 diameters. 3. Results
All of the hearts of the control mice were normal and free of viral crystals. The myocardium of the control mice appeared as previously described [ 121. The damage to the myocardium of the infected animals ranged from mild to severe and varied with the area of the heart examined. Where viral crystals were located, the damage was usually moderate to severe. Viral crystals were easily found in the myocardium of 31 of the newborn mice (Plates 1 to 3), but crystals were not found in the 8-day-old mice. A definite infection with EMC. virus could easily be ascertained in those mice in which viral crystals were found. In the myocardium of other mice, lesionsranging from mild to severe were also observed. The fact that viral crystals were not found in somemice was not unexpected since the area of tissueexamined with the electron
PI,ATE I. Myocardium \ irus ct~lturc HIrid. Notr the thr viral cryd f\T). In one g:lycwg~~~~ p;rrticlr~ CC;) can bv
of a newborn mouse killed one day after membrane-bound vrsiclrs and vacuoles vcsiclc (big arrow) remnants of cytoplasm obacrved. The cristav of the mitochondria
inoculation with 0.1 ml EhlC (small arrows) associated with can br seen. In othw vcsiclra arc slightly swollrr~. 52 000.
PLATE 2. Myocardium of a newborn mouse killed one day following EMC virus culture fluid. Note the viral crystal (V) associated with a minor are organized into a cubic configuration. x 52 000.
inoculation with 0.1 ml lesion. The viral particles
PLh’l‘E 3. Myocardium of a newborn mouse killed one day virus culture fluid. A large portion of the cytoplasm is occupird L acuolrs. A viral crystal (V) is present. There are no myofibrils. (listorted. 3fi 400.
after inoculation with 0.05 ml EhlC by membrane-bound vesicles and The mitochondria (hl) arv ,grosA
PLATE 4. Myocardium of an &day-old mouse killed one day after inoculation virus culture fluid, showing a widespread lesion which extends to the periphery capillary containing a red blood cell (RBC) can be sem. x 26 000.
with 0.05 ml EMC of the myocyte. A
PLATE 5. Myocardium of a newborn mouse killed one day after inoculation with 0.05 ml CHIC’ virus culture fluid. A sharply isolated necrotic area of cytoplasm is located within otherwise wl;ltiwl\ normal myoplasm. The mitochondria (M) in the necrotic arca arc enlarged. .. 36 400.
CYTOPATHOLOGY
OF EMC-INFECTED
MOUSE
MYOCARDIUM
31
microscope is extremely small and #therefore viral crystals could be present in the myocardium but not noted in the sections focused upon. The damage to the myocardium was focal. In some areas the myocardium appeared relatively normal, whereas in other areas the damage appeared moderate to severe. Lesions of one or several myocardial cells were found in areas where most of the myocytes appeared normal The most characteristic feature of the lesions caused by the in vivo infection with EMC virus was the presence of membrane-bound vesicles and vacuoles. In some areas, these were few in number; in other areas the cytoplasm was entirely filled with these vesicles and vacuoles. Some of these vesicles contained what appeared to be remnants of cytoplasm. In other vesicles glycogen granules appeared (Plate 1). Some of the vesicles and vacuoles had a dense membrane. In these lesions, viral crystals were frequently encountered (Plates 1 and 2). Also, in these lesions the mitochondria were enlarged, the cristae swollen, and the myofibrils disorganized. In the less damaged areas, vesicles and vacuoles were frequently found scattered among the mitochondria which were intact except that the cristae were somewhat dilated (Plate 1). In some cells the cytoplasmic lesion occupied only a small portion of the cytoplasm (Plates 1 and 2), whereas in other cells the lesions were more extensive and occupied or destroyed the entire cytoplasm (Plates 3 and 4). When the lesions were minor they were usually located in the central portion of the cell, but in some of the more extensive lesions, the necrosis extended to the periphery of the cell (Plate 4). Where the vesicles and vacuoles were most numerous, the myoplasm was completely lacking and no myofibrils could be recognized (Plate 3), the sarcoplasmic reticulum and T-system were lacking, and the mitochondria were grossly distorted. In some areas, portions of necrotic cytoplasm appeared to be isolated or separated from the relatively normal myocardium (Plate 5). In such damaged areas the mitochondria were enlarged, and membranous cytoplasmic remnants were present. 4. Discussion The lesions of the myocardium described above were fairly consistently found in EMC virus infected mice. Somewhat simiIar lesions were found in the cardiac valves [II], pancreas [15] and kidney [18] of EMC virus infected mice. Similar cytopathic changes have been described for various types of tissue culture cells infected with various viruses [ZZ, 32, 391. In vitro studies with poliovirus [21] and Mengovirus [2, 31 have shown similar changes. In these in vitro studies, the changes consisted of the formation of membrane-bound vesicles and vacuoles within the cytoplasm and were often found in association with viral crystals. We found definite viral crystals in the myocardium of 31 of the 39 newborn infected animals studied. It is likely that viral crystals were present in the myo-
58
J. M. HARB
AND
G. E. BURGH
cardium of the other newborn mice but were not observed because the electron microscopic sections include extremely small portions of the myocardium. Why viral crystals were not found in any of the 8-day-old infected animals is not known. However, the fact that newborn mice are highly susceptible to EMC viral infection whereas older mice are not may explain why the crystals were readily found in the former but not in the latter animals. Nevertheless, similar cytopathic changes were observed in both newborn and 8-day-old mice. In the mice in which lesions were found but in which viral crystals were not located (both newborn and 8-day-old mice), the lesions were most likely the result of viral invasion of the myocardium. The viral crystals were readily distinguished from glycogen granules and free ribosomes because of the difference in size of the particles and because the viral particles stained more intensely with uranyl and lead salts than the glycogen granules or ribosomes. Furthermore, particles of the viral crystals were organized into cubic arrangements (Plates 2 and 3) by which they were easily distinguished from the randomly scattered glycogen particles and free ribosomes. In these experiments with mice, it appears there was marked proliferation of the cytoplasmic membrane systems, as evidenced by the large amount of membrane material in the more severe and extensive lesions. It has been shown previously [21] that a characteristic cellular response to in vitro infection with picornaviruses is a rapid proliferation of numerous membrane enveloped cisternae. The demonstration [3] of enhanced incorporation of radioactive choline in Mengovirus infected cells indicates an enhanced synthesis of membrane material, supporting the observation of proliferation of the cytoplasmic membrane systems following viral infection. Two questions, therefore, remain: (i) from whence are the membranes formed; and (ii) what is the mechanism which stimulates membrane proliferation? Neither the sarcoplasmic reticulum nor the transverse tubular system could be recognized in the lesions observed here. The membranes from these various systems, including the membranes of degenerated mitochondria, could account for some of the membrane material associated with the formation of the numerous vesicles and vacuoles observed. In studies by others in which cells were incubated in radioactive choline prior to infection with Mengovirus, the tracer was found to be concentrated in the vicinity of newly formed cisternae, thus implying that some of the lipid contained in these newly assembled membranes was derived from pre-existent macromolecules bound in cellular lipids [3]. In addition, when cells were incubated in radioactive choline following infection with Mengovirus, it was shown that choline was incorporated into the smooth membrane cisternae which proliferated after infection. Thus, in experiments with cells labeled before and after infection, it has been shown that phospholipid that is incorporated in new membranes can be derived from pre-existing macromolecules and newly synthesized membranes
c31* The question is still unanswered as to what cytotoxic factor causes membrane proliferation and ultimate lysis of the cell. One theory is that the cytotoxic factor
CYTOPATHOLOGY
OF EMC-INFECTED
MOUSE
MYOCARDIUM
59
is a viral protein. This protein could be the viral coat or a protein synthesized by the virus. It has been shown that cytopathic effects result as a late viral function, at a time when synthesis of coat protein is rapid [Z]. Another theory is that the virus causes a leakage of lysosomal hydrolases, which results in cellular disruption and lysis. It has been demonstrated that lysosomal hydrolases are released following infection with picornaviruses [1, 271. In support of this second theory, it has been shown [26] that the synthesis of phospholipids is increased in the presence of lysolecithin, a product of the hydrolysis of lipid. On the other hand, when hydrocortisone, a known stabilizer of lysosomal membranes, is added to the nutrient medium prior to virus infection, cytopathic effects are delayed [2]. Furthermore, hydrocortisone suppresses the incorporation of choline in both infected and control cells [3]. It appears, therefore, that the stimulation of membrane proliferation is caused by a release of lysosomal enzymes into the cytoplasm. It is most probable that this release of enzymes is initiated as a cellular response to a protein synthesized by the virus, probably during its replicative stage. In response to the released hydrolases, new membranes are formed which surround fragments of cytoplasm. There is a strong correlation between these membrane structures and enhanced synthesis of lipid. Lysolecithin, a degradative product of lysosomal hydrolysis, could cause a breakdown and turnover of pre-existing lipids as well as a stimulation of phospholipid synthesis [26]. The degree of damage to some myocytes was so extreme that the myocytes could not function in contraction. In most damaged myocytes the myofibrils were disorganized and reduced in number. In addition, other cytoplasmic systems, such as mitochondria, sarcoplasmic reticulum and the transverse tubular system, were altered or destroyed. In the absence of these organelles normal contraction could not be expected. We conclude that the cytoplasmic effects of EMC virus on the myocardium of mice reported here were the result of direct viral infection of the myocardium per se. Not all of the myocytes were affected, but in those that did show cytopathology, the lesions were fairly consistent in character.
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A. C. & SANDELIN, K. Activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects. 3ournal of Ex@nhental Medicine 117, 879-887 (1963). AMAKO, K. & DALES, S. Cytopathology of Mengovirus infection. I. Relationship between cellular disintegration and virulence. Virology 32, 184-200 (1967). AMAKA, K. & DALES, S. Cytopathology of Mengovirus infection. II. Proliferation of membranous cisternae. Virology 32, 201-2 15 (1967). BWLOCK, Z. R., RABIN, E. R. & MELNICK, J. L. Adenovirus endocarditis in mice. Science 157, 69-70 (1967). ALLISON,
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BLAILOCK, Z. R., RABIN, E. R. & MELNICK, J. L. Adenovirus myocarditis in mice: An electron microscopic study. Experimental and Molecular Pathology 9, 84-96 (1968). 6. BURCH, G. E. & COLCOLOUGH, H. L. Viral valvulitis. American Heart Journal 78, 119123 (1969). 7. BURGH, G. E. & COLCOLOUGH, H. L. Progressive Coxsackie viral pancarditis and nephritis. Annals of Internal Medicine 71, 963-970 (1969). 8. BURCH, G. E. & DEPASQUALE, N. P. Viral endocarditis. American Heart Journal 67,72 l723 (1964). 9. BURGH, G. E., DEPASQUALE, N. P., SUN, S. C., HALE, A. R. & MOGABGAB, W.J. Experimental Coxsackie virus endocarditis. 77~ Journal of the American Medical Association 196, 349-352 (1966). 10. BURGH, G. E., DEPASQUALE, N. P., SUN, S. C., MOGABGAB, W. J. & HALE, A. R. Endocarditis in mice infected with Coxsackie virus Bq. Science 151, 447-448 (1966). Il. BURCH, G. E. & tinn, J. M. Encephalomyocarditis viral valvulitis in new-born mouse. Exjwrientia
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BURGH, G. E., HARB, J. M., COLCOLOUGH, H. L. & Tsur, C. Y. Encephalomyocarditis infection of the newborn mouse myocardium: An electron microscopic study. Archives of Internal Medicine 127, 148-156 (1971). 13. BURCH,G. E., SUN, S. C., COLCOLOUGH, H. L., SOHAL, R. S. & DEPASQUALE,N. P. Coxsackie virus valvulitis and myocarditis observed at routine autopsy. Exfierentia 23, l-5 (1967). 14. BURCH, G. E., TSUI, C. Y. & HARB, J. M. Pathologic changes of aorta and coronary arteries of mice infected with Coxsackie B4 virus. Proceedings of the Societyfor Expimental Biology and Medicine 137, 657-661 (1971). 15. BURGH, G. E., Tsur, C. Y. & HARB, J. M. Pancreatitis of mice infected with encephalomyocarditis (EMC) virus. In preparation. 16. BIJRCH, G. E., Tsur, C. Y. & HARB, J. M. Pancreatic islet cell damage in mice produced by Coxsackie Bi and encephalomyocarditis viruses. Experientia, in press. 17. BURCH, G. E., TSUI, C. Y. & HARB, J. M. Hepatitis in mice infected with Coxsackievirus BI. In preparation. 18. BURCH, G. E., Tsut, C. Y. & HARB, J. M. The early renal lesions of mice infected with encephalomyocarditis virus. Laboratory Investigation, in press. 19. BURCH, G. E., TSUI, C. Y., HARB, J. M. & COLCOLOUGH, H. L. Mural and valvular endocarditis of mice infected with encephalomyocarditis (EMC) virus. Experimental and Molecular Pathology 14, 327-336 (1971). 20. BURCH, G. E., TSUI, C. Y., HARB, J. M. & COLCOLOUGH, H. L. Pathologic findings in the pancreas of mice infected with Coxsackie virus B4. Archives of Internal Medicine 128, 40-47 (1971). 21. DALES, S., EGGERS, H. J., TAMM, I. & PALADE, G. E. Electron microscopic study of the formation of poliovirus. Virolog 26, 379-389 (1965). 22. DALES, S. & FRANKLIN, R. M. A comparison of the changes in fine structure of L cells during single cycles of viral multiplication, following their infection with the TheJournal of Cell Biology 14, 281-302 viruses of Mengo and encephalomyocarditis. (1962). 23. DEPASQUALE, N. P., BURGH, G. E., SUN, S. C., HYALE, A. R. & MOGABGAB, W.J. Experimental Coxsackie virus B4 valvulitis in cynomolgus monkeys. American Heart 3ourml71, 678-683 (1966). 24. DICK, G. W. A., SMITHBURN, K. C. & -DOW, A. J. Mengo encephalomyelitis virus: Isolation and immunological properties. British Journal of Exferimental Pathology 29, 547-558 ( 1948).
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FINDLAY, G. M. & HOWARD, E. M. Observations of Pathology and Bacteriology 63, 435-443. (1951). FISCUS, W. G. & SCHNEIDER, W. C. The role phorylcholine (1966).
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39.
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cytidyltransferase
activity.
Journal
on Columbia
SK virus.
61 TheJournal
of phospholipids in stimulating phosof Biological Chemistry 241, 3324-3330
FLANAGAN, J. F. Hydrolytic enzymes in KB cells infected simplex virus. Journal of Bacteriology 91, 789-797 (1966). GAINER, J. H. & MURCHISON, T. E. Encephalomyocarditis
with
poliovirus
and
herpes
virus infection of swine. Medicine 56, 173-175 (1961). HASSAN, S. A., RABIN, E. R. & MELNICK, J. L. Reovirus myocarditis in mice: An electron microscopic, immunofluorescent and virus assay study. Experimental and Molecular Pathology 4, 6680 (1965). HELWIG, F. C. & SCHMIDT, E. C. H. A filter-passing agent producing interstitial myocarditis in anthropoid apes and small animals. Science 102, 31-33 (1945). JUNGEBLUT, C. W., FEINER, R. R. & SANDERS, M. Studies in rodent poliomyelitis: III. Experimental poliomyelitis in Guinea pigs produced with the murine strain of SK poliomyelitis virus. Journal of Experimental Medicine 76, 3 l-51 (1942). KALLMAN, F., WILLIAMS, R. C., DULBECCO, R. & VOGT, M. Fine structure of changes produced in cultured cells sampled at specified intervals during a single growth cycle of polio virus. lT& Journal of Biophysical and Biochemical Cytology 4, 301-308 (1958). KILHAM, L., MASON, P. & DAVIES, J. N. P. Host-virus relations in encephalomyocard&is (EMC) virus infections: I. Infections of wild rats. American Journal of Tropical Medicine and Hygiene 5, 647-654 (1956). KILHAM, L., MASON, P. & DAVIES, J. N. P. Host-virus relations in encephalomyocarditis (EMC) virus infections: II. Myocarditis in mongooses. American Journal oJ Tropical Medicine and Hygiene 5, 655-663 (1956). MURNANE, T.G., CRAIGHEAD, J.E., MONDRAGON, H. & SHELOKOV, A.Fataldiseaseof swine due to encephalomyocarditis virus. Science 131, 498-499 (1960). POPE, J. H. A virus of the encephalomyocarditis group from a water-rat, Hydromys Chrysogaster, in North Queensland. Australian Journal of Experimental Biology and Medical Science 37, 117-124 (1959). RABIN, E. R., HASSAN, S. A., JENSON, A. B. & MELNICK, J. L. Coxsackie virus Bs myocarditis in mice: An electron microscopic, immunofluorescent and virus-assay study. The American journal of Pathology 44, 775-797 (1964). RABIN, E. R., PHILLIPS, C. A., JENSON, A. B. & MELNICK, J. L. Vaccinia virus myocarditis in mice: An electron microscopic and virus assay study. Experimental and Molecular Pathology 4, 98-l 11 (1965). RIPKIND, R. A., GODMAN, G. C., Hewn, C., MORGAN, C. & ROSE, H. M. Structure and development of viruses as observed in the electron microscope. VI. ECHO virus, type 9. Journal of Experimental Medicine 114, 1-12 (1961). ROCA-GARCIA, M. & SANMARTIN-BARBERI, C. The isoIation of encephalomyocarditis virus from Aotus monkeys. American Journal of Tropical Medicine and Hygiene 6, 840-852 (1957). SCHMIDT, E. C. H. Virus myocarditis: pathologic and experimental studies. 77ze American Journal of Pathology 24, 97-l 17 ( 1948). SOHAL, R. S. & BURGH, G. E. Electron microscopic study of the endocardium in Coxsackie virus B4 infected mice. The American3ournal of Pathology 55, 133-145 (1969). SUN, S. C., COLCOLOUGH, H. L., BURCH, G. E., DEPASQUALE, N. P. & SOHAL, R. S. Immunofluorescent study of B4 Coxsackievirus valvulitis in mice. Proceedings of the Society,for Experimental Biology and Medicine 125, 157-162 (1967). Veterinarian
29.
OF EMC-INFECTED MOUSE MYOCARDIUM
62 44. 45.
46. 47. 48.
J.M. HARB AND G.E.BURCH Sm,S. C.,SOHAL, R.S., BURCH,G. E., CHU, K. C. & COLCOLOUGH,H.L.COXS~~& virus B4 pancarditis in cynomolgus monkeys resembling rheumatic heart lesions. British 3ournal of Ex@bnental Pathology 48, 655-661 (1961). SUN,S.C.,SOHAL, R.S., CHU, K.C., COLCOLOUGH, H.L., LEIDERMAN, E. &BURCH, G. E. Mycoplasma gallisepticum infection in mice and cynomolgus monkeys: A histologic, immunofluorescence, and electron microscopic study of the heart. 77u American 3oumzl of Pathology 53, 1073-1096 (1968). TSUI, C. Y. & BURCH, G. E. Coxsackie virus B4 pericarditis in mice. British Journal of Experimental Pathology 52, 47-50 (197 1). TSUI, C. Y., BURCH, G. E., COLCOLOUGH, H. L. & b, J. M. Early myocardial lesions in encephalomyocarditis (EMC) virus infected mice. Cardiovascular Research 5, 550-557 (1971). Tsm, C. Y., BURCH, G. E. & m, J. M. Pancreatitis in mice infected with Coxsackie virus Br. Archives of Pathology, in press.
and Cellular Cardiology
(1973)
5,55-62
Ultrastructural Cytopathology Associated with EMC
Department
of Medicine
of Mouse Myocardium Viral Infection*
J. M. HARB AND G. E. BURGH of the Tulane University School of Medicine Louisiana, (Received
22 May
.New Orleans,
Louisiana,
and th
Charity
Hospital
of
U.S.A.
1972, and accepted 24 August
1972)
J. M. HARB AND G. E. BURGH Ultrastructural Cytopathology of Mouse Myocardium Associated with EMC Viral Infection. Journal of Molecular and Cellular Cardiology (1973) 5, 5562. Lesions in the mouse myocardium are described which are fairly characteristic of cytopathology caused by direct infection with EMC virus. The lesions consisted of formation of numerous cytoplasmic membrane-bound vesicles and vacuoles. Viral crystals were frequently associated with these lesions in the newborn mouse. Accompanying the lesion was a breakdown of the myofibrils, mitochondria, sarcoplasmic reticulum and transverse tubular system. The absence of these organelks clearly indicates a reduced contractability of the damaged myocytes. Possible factors resulting in membrane proliferation and mechanisms for cell lysis are discussed.
1. Introduction We have described experimental infections of mice with encephalomyocarditis (EMC) virus, Coxsackie viruses B1 and B4, ECHO 9 virus and mycoplasma gallisepticum agent [453. These studies have included investigations of the damage caused by viral infection to the myocardium [ 22, 471, endocardium [8], valves [II, 19, 231, aorta [14], coronary arteries [14], pancreas [15, 16, 20, 481, liver [17] and kidneys [ 281. It has been shown that myocarditis can be produced by many viruses, including reovirus [29], vaccinia virus [38], adenovirus [S], and Coxsackie virus Bs [37j, as well as those mentioned above. Viral damage to the heart may also appear as mural endocarditis [4, 9, 10, 19, 421, valvulitis [S, II, 13, 23, 431, pericarditis [46], or pancarditis [7, 441. The EMC virus, as used in this study, has been shown to cause myocardial damage in a number of animals [IZ, 24, 25, 28, 31, 33, 34, 35, 36, 411. The EMC virus was initially recovered from anthropoid apes [30] and was later isolated also from Aotus monkeys [40]. In our studies of EMC virus infections of the myocardium of the mouse [12], we noted a peculiar and somewhat characteristic ultrastructural cytopathology * Supported by grant HE-06769 from the National Heart and Lung Institute of the U.S. Health Service, the Rudolph Matas Memorial Fund for the Kate Prewitt Hess Laboratory, Rowe11 A. Billups Fund for Research in Heart Disease and the Feazel Laboratory
Public the
56
J. hf. HARB
AND
G. E. BURGH
associated with the formation of viral crystals. It is the purpose of this paper to report and to describe this cytopathology in detail.
2. Methods
and
Materials
A total of 39 newborn and 15 S-day-old Swissmice were inoculated with 0.05 ml or 0.1 ml of EMC virus culture fluid with a titre of 10-e TCID~(I. Thirteen control Swissmice (nine newborn and four 8-day-old animals) were inoculated with 0.1 ml of virus-free culture fluid. The mice were killed 24 h after inoculation, except for one control mouse which was killed 2 days after inoculation. Five of the infected newborn mice were found dead in their cages 24 h after inoculation. These five mice were included in this study along with the others. A portion of the left ventricle was excised immediately after the mice were killed by cervical spinal dislocation. The tissueswere minced into 1 mm3 pieces in a pool of cold 3% phosphate-buffered glutaraldehyde. The piecesof tissuewere then placed in fresh glutaraldehyde for 2 h. Following a 3-h rinse with 6 to 8 changes of fresh phosphate buffer, the tissueswere post-fixed for 13 h in cold 1o/ophosphatebuffered osmium tetroxide. Dehydration was accomplished in a graded series of alcohol. After infiltration with propylene oxide, the tissueswere placed in a mixture of 3 propylene oxide and JJepon and left under a lamp overnight. The tissues were then put through two l-h changes of pure epon and were finally embedded in preformed capsules. The tissueswere allowed to cure in an oven at 60°C for 72 h. Thin sections (400 to 600 A) were cut using both glass and diamond knives. The sectionswere stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop I electron microscope at initial magnifications of 10 000, 14 000 and 18 000 diameters. Viral crystals were readily visible at a magnification of 14 000 diameters. 3. Results
All of the hearts of the control mice were normal and free of viral crystals. The myocardium of the control mice appeared as previously described [ 121. The damage to the myocardium of the infected animals ranged from mild to severe and varied with the area of the heart examined. Where viral crystals were located, the damage was usually moderate to severe. Viral crystals were easily found in the myocardium of 31 of the newborn mice (Plates 1 to 3), but crystals were not found in the 8-day-old mice. A definite infection with EMC. virus could easily be ascertained in those mice in which viral crystals were found. In the myocardium of other mice, lesionsranging from mild to severe were also observed. The fact that viral crystals were not found in somemice was not unexpected since the area of tissueexamined with the electron
PI,ATE I. Myocardium \ irus ct~lturc HIrid. Notr the thr viral cryd f\T). In one g:lycwg~~~~ p;rrticlr~ CC;) can bv
of a newborn mouse killed one day after membrane-bound vrsiclrs and vacuoles vcsiclc (big arrow) remnants of cytoplasm obacrved. The cristav of the mitochondria
inoculation with 0.1 ml EhlC (small arrows) associated with can br seen. In othw vcsiclra arc slightly swollrr~. 52 000.
PLATE 2. Myocardium of a newborn mouse killed one day following EMC virus culture fluid. Note the viral crystal (V) associated with a minor are organized into a cubic configuration. x 52 000.
inoculation with 0.1 ml lesion. The viral particles
PLh’l‘E 3. Myocardium of a newborn mouse killed one day virus culture fluid. A large portion of the cytoplasm is occupird L acuolrs. A viral crystal (V) is present. There are no myofibrils. (listorted. 3fi 400.
after inoculation with 0.05 ml EhlC by membrane-bound vesicles and The mitochondria (hl) arv ,grosA
PLATE 4. Myocardium of an &day-old mouse killed one day after inoculation virus culture fluid, showing a widespread lesion which extends to the periphery capillary containing a red blood cell (RBC) can be sem. x 26 000.
with 0.05 ml EMC of the myocyte. A
PLATE 5. Myocardium of a newborn mouse killed one day after inoculation with 0.05 ml CHIC’ virus culture fluid. A sharply isolated necrotic area of cytoplasm is located within otherwise wl;ltiwl\ normal myoplasm. The mitochondria (M) in the necrotic arca arc enlarged. .. 36 400.
CYTOPATHOLOGY
OF EMC-INFECTED
MOUSE
MYOCARDIUM
31
microscope is extremely small and #therefore viral crystals could be present in the myocardium but not noted in the sections focused upon. The damage to the myocardium was focal. In some areas the myocardium appeared relatively normal, whereas in other areas the damage appeared moderate to severe. Lesions of one or several myocardial cells were found in areas where most of the myocytes appeared normal The most characteristic feature of the lesions caused by the in vivo infection with EMC virus was the presence of membrane-bound vesicles and vacuoles. In some areas, these were few in number; in other areas the cytoplasm was entirely filled with these vesicles and vacuoles. Some of these vesicles contained what appeared to be remnants of cytoplasm. In other vesicles glycogen granules appeared (Plate 1). Some of the vesicles and vacuoles had a dense membrane. In these lesions, viral crystals were frequently encountered (Plates 1 and 2). Also, in these lesions the mitochondria were enlarged, the cristae swollen, and the myofibrils disorganized. In the less damaged areas, vesicles and vacuoles were frequently found scattered among the mitochondria which were intact except that the cristae were somewhat dilated (Plate 1). In some cells the cytoplasmic lesion occupied only a small portion of the cytoplasm (Plates 1 and 2), whereas in other cells the lesions were more extensive and occupied or destroyed the entire cytoplasm (Plates 3 and 4). When the lesions were minor they were usually located in the central portion of the cell, but in some of the more extensive lesions, the necrosis extended to the periphery of the cell (Plate 4). Where the vesicles and vacuoles were most numerous, the myoplasm was completely lacking and no myofibrils could be recognized (Plate 3), the sarcoplasmic reticulum and T-system were lacking, and the mitochondria were grossly distorted. In some areas, portions of necrotic cytoplasm appeared to be isolated or separated from the relatively normal myocardium (Plate 5). In such damaged areas the mitochondria were enlarged, and membranous cytoplasmic remnants were present. 4. Discussion The lesions of the myocardium described above were fairly consistently found in EMC virus infected mice. Somewhat simiIar lesions were found in the cardiac valves [II], pancreas [15] and kidney [18] of EMC virus infected mice. Similar cytopathic changes have been described for various types of tissue culture cells infected with various viruses [ZZ, 32, 391. In vitro studies with poliovirus [21] and Mengovirus [2, 31 have shown similar changes. In these in vitro studies, the changes consisted of the formation of membrane-bound vesicles and vacuoles within the cytoplasm and were often found in association with viral crystals. We found definite viral crystals in the myocardium of 31 of the 39 newborn infected animals studied. It is likely that viral crystals were present in the myo-
58
J. M. HARB
AND
G. E. BURGH
cardium of the other newborn mice but were not observed because the electron microscopic sections include extremely small portions of the myocardium. Why viral crystals were not found in any of the 8-day-old infected animals is not known. However, the fact that newborn mice are highly susceptible to EMC viral infection whereas older mice are not may explain why the crystals were readily found in the former but not in the latter animals. Nevertheless, similar cytopathic changes were observed in both newborn and 8-day-old mice. In the mice in which lesions were found but in which viral crystals were not located (both newborn and 8-day-old mice), the lesions were most likely the result of viral invasion of the myocardium. The viral crystals were readily distinguished from glycogen granules and free ribosomes because of the difference in size of the particles and because the viral particles stained more intensely with uranyl and lead salts than the glycogen granules or ribosomes. Furthermore, particles of the viral crystals were organized into cubic arrangements (Plates 2 and 3) by which they were easily distinguished from the randomly scattered glycogen particles and free ribosomes. In these experiments with mice, it appears there was marked proliferation of the cytoplasmic membrane systems, as evidenced by the large amount of membrane material in the more severe and extensive lesions. It has been shown previously [21] that a characteristic cellular response to in vitro infection with picornaviruses is a rapid proliferation of numerous membrane enveloped cisternae. The demonstration [3] of enhanced incorporation of radioactive choline in Mengovirus infected cells indicates an enhanced synthesis of membrane material, supporting the observation of proliferation of the cytoplasmic membrane systems following viral infection. Two questions, therefore, remain: (i) from whence are the membranes formed; and (ii) what is the mechanism which stimulates membrane proliferation? Neither the sarcoplasmic reticulum nor the transverse tubular system could be recognized in the lesions observed here. The membranes from these various systems, including the membranes of degenerated mitochondria, could account for some of the membrane material associated with the formation of the numerous vesicles and vacuoles observed. In studies by others in which cells were incubated in radioactive choline prior to infection with Mengovirus, the tracer was found to be concentrated in the vicinity of newly formed cisternae, thus implying that some of the lipid contained in these newly assembled membranes was derived from pre-existent macromolecules bound in cellular lipids [3]. In addition, when cells were incubated in radioactive choline following infection with Mengovirus, it was shown that choline was incorporated into the smooth membrane cisternae which proliferated after infection. Thus, in experiments with cells labeled before and after infection, it has been shown that phospholipid that is incorporated in new membranes can be derived from pre-existing macromolecules and newly synthesized membranes
c31* The question is still unanswered as to what cytotoxic factor causes membrane proliferation and ultimate lysis of the cell. One theory is that the cytotoxic factor
CYTOPATHOLOGY
OF EMC-INFECTED
MOUSE
MYOCARDIUM
59
is a viral protein. This protein could be the viral coat or a protein synthesized by the virus. It has been shown that cytopathic effects result as a late viral function, at a time when synthesis of coat protein is rapid [Z]. Another theory is that the virus causes a leakage of lysosomal hydrolases, which results in cellular disruption and lysis. It has been demonstrated that lysosomal hydrolases are released following infection with picornaviruses [1, 271. In support of this second theory, it has been shown [26] that the synthesis of phospholipids is increased in the presence of lysolecithin, a product of the hydrolysis of lipid. On the other hand, when hydrocortisone, a known stabilizer of lysosomal membranes, is added to the nutrient medium prior to virus infection, cytopathic effects are delayed [2]. Furthermore, hydrocortisone suppresses the incorporation of choline in both infected and control cells [3]. It appears, therefore, that the stimulation of membrane proliferation is caused by a release of lysosomal enzymes into the cytoplasm. It is most probable that this release of enzymes is initiated as a cellular response to a protein synthesized by the virus, probably during its replicative stage. In response to the released hydrolases, new membranes are formed which surround fragments of cytoplasm. There is a strong correlation between these membrane structures and enhanced synthesis of lipid. Lysolecithin, a degradative product of lysosomal hydrolysis, could cause a breakdown and turnover of pre-existing lipids as well as a stimulation of phospholipid synthesis [26]. The degree of damage to some myocytes was so extreme that the myocytes could not function in contraction. In most damaged myocytes the myofibrils were disorganized and reduced in number. In addition, other cytoplasmic systems, such as mitochondria, sarcoplasmic reticulum and the transverse tubular system, were altered or destroyed. In the absence of these organelles normal contraction could not be expected. We conclude that the cytoplasmic effects of EMC virus on the myocardium of mice reported here were the result of direct viral infection of the myocardium per se. Not all of the myocytes were affected, but in those that did show cytopathology, the lesions were fairly consistent in character.
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