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Experimental Coxsackie virus B4 valvulitis in cynomolgus monkeys
Experimental Cox__ie virus 1 4 valvulitis in cynomeltfusmonkeys Nicholas P. DePasquale, lvI.D. George E. Burch, ltf.D.* Shih C. Sun, M.D. Alfred R. Hale, M.D. William J. Mogabgab, M.D. New Orleans, La.
A lthough it is known that many viruses ~ produce myocarditis and pericarditis in man,1-5 endocarditis is not generally considered to be a complication of viral infection. 6 Studies in this laboratory have shown that mural and valvular endocarditis can be produced consistently in mice with Coxsackie virus B 4.7 Because of the implications of these findings relative to the etiology of valvular heart disease in man the present study in which Coxsackie virus infection was induced in primates (cynomolgus monkeys) was performed. This paper describes the valvular lesions produced in cynomolgus monkeys with Coxsackie virus B 4 • Material and methods Virus stock. The Coxsackie virus B 4 used in these experiments was originally recovered in 1958, by Kibrick and Benirschke from a lO-day-old infant who died of encephalohepatomyocarditis. 8 The virus obtained as monkey kidney culture passage strain was prepared in rhesus monkey kidney culture. 9 Control fluid from virus-
free monkey kidney culture was also obtained. Virus and control fluid was stored at -65°C. Monkeys. Nine young adult cynomolgus monkeys were obtained from Asiatic Animal Imports, San Francisco, California. Inoculation of virus and collection of tissue. Seven monkeys were inoculated intravenously with 0.3 m!. of monkey kidney culture fluid containing Coxsackie virus B 4 105TCID 5o . The monkeys were killed 51 to 200 days after inoculation. Control experiments. Two monkeys were inoculated intravenously with 0.3 m!. of virus-free monkey kidney culture fluid. One monkey was killed 51 days after inoculation, and the other, 231 days after inoculation. Histologic studies. Serial sections of pericardium heart muscle, endocardium, and valves were stained with hematoxylin and eosin. Direct fluorescent antibody staining 10 was used to identify viral antigen in the tissues in 3 of the virus-infected animals and in 1 of the control animals.
From the Department of Medicine, Tulane University School of Medicine, and Charity Hospital of Louisiana, New Orleans, La. Supported by grants from the United States Public Health Service. Received for publication Jan. 11, 1966. *Address: Department of Medicine, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans. La., 70112.
678
Volume 71 Number 5
Coxsackie virus B 4 valvulitis in cynomolgus monkeys
Results Valvulitis was found in 6 of the 7 monkeys inoculated with Coxsackie virus B 4 and in neither of the 2 control animals (Table I). Grossly, the lesions consisted of (1) verrucous aortic valvulitis in 2 monkeys, (2) verrucous mitral valvulitis in 3 monkeys (Fig. 1), and (3) cicatricial thickening of the mitral valve leaflets and chordae tendineae with commissural adhesions (stenosis) in 2 monkeys (Fig. 2).
Table 1. Valve lesions in cynomolgus monkeys inoculated with Coxsackie virus B 4 Animal number*
Days infected
1. 5.
51 51 74
6. 7.
178 185
8. 9.
200
2.
199
Valve lesion Verrucous lesion of mitral valve Verrucous lesion of aortic valve Verrucous lesions of aortic and mitral valve Verrucous lesion of mitral valve Thick, scarred mitral valve with commissural adhesions None Thick, scarred mitral valve with commissural adhesions
*N 0 valve lesions were found in control cynomolgus monkeys Nos. 3 and 4.
679
Histologically, the valve tissue from the infected animals displayed stromal edema, round cell infiltration, fibrocytic proliferation, increased basophilia, and swelling of endothelial cells (Figs. 3, 4, and 5). In the 3 virus-inoculated animals in which direct fluorescent antibody staining was carried out, viral antigen was identified in the tissues (Fig. 6), whereas fluorescent antibody staining was negative in the single control animal in which this technique was employed. Attempts to recover Co-xsackie virus from the tissues of the infected animals were unsuccessful.
Discussion Lou, Wenner and Kamitsuka,ll using the same strain of Coxsackie virus as that employed in the present studies, found mural endocarditis in 4, and mitral valvulitis in 2, of 9 cynomolgus monkeys inoculated with the virus. The valvular lesions described by Lou and associates consisted primarily of infiltration with polymorphonuclear neutrophils and swelling of the endothelial lining cells. Although fibrocytic proliferation and commissural adhesions were not described, these workers killed the monkeys within 28 days of
Fig. 1. Photograph showing verrucous lesion of the mitral valve associated with thickening of the valve in a cynomolgus monkey inoculated with Coxsackie virus B 4 178 days before autopsy.
680
DePasquale, Burch, Sun, Hale, and
JJo.~abgab
~Jm> /-feart .! ;HO)',l f}(j/',
Fig. 2. Photograph showing tlbrosis and thickening of the mitral valve associated with commissural adhesions (stenosis) and thickening and shortening of the chordae tendineae in a cyllolllolgns monkE'v inoculated with Coxsackie virus B, 200 days before autopsy.
inoculation. Thus, the valvular lesions observed by Lou and associates were more acute than those described in the present report. It is of particular interest that valvular lesions similar to those of human mitral stenosis with commissural adhesion and thickening and shortening of the chordae tendineae and papillary muscles were observed (Fig. 2) in 2 animals in our series. In spite of the fact that attempts to recover virus from the tissues were unsuccessful, viral antigen was identified in the valves for as long as 200 days after inoculation (Fig. 6). In addition to the present studies and those of Lou and associates, viral endocarditis has been produced experimentally in mammals by Pearce,12 using Virus III in rabbits, and by Kilham, Mason and Davies,13 using encephalomyocarditis virus in mongooses. In addition, Karsner 14 and Tedeschi and Stevenson 15 have described instances of valvulitis in man which were probably viral in etiology. The present studies, as well as those of Lou, Wenner and Kamitsuka, demonstrate that valvulitis can be produced in cynomolgus monkeys with Coxsackie virus B 4 • Since Coxsackie viruses are among the
most common infective agents of man,16 it would be important to know whether these viruses produce valvular lesions in man similar to those produced in monkeys. Valvular lesions were not described in pathologic material from several epidemics of Coxsackie virus myocarditis among newborn infantsY-19 However, in none of the infants who died during these epidemics were histologic studies of the valves described. Since death usually occurred within a week to 10 days of birth, sufficient time may not have elapsed to produce gross valvular lesions. In adults, Coxsackie virus infection of the heart is not usually fatal, so that the opportunity to study pathologic material is limited. However, murmurs have been described which developed during acute Coxsackie virus myocarditis and persisted long after the infection had subsided. 20 It is well known that many patients with aortic and/or mitral valvular disease give no history of rheumatic fever. Nevertheless, the clinician is usually satisfied to render a diagnosis of rheumatic valvulitis if no other cause of the valvular lesion can be found. We think that some instances of chronic valvular disease may be due to Coxsackie virus infection. It is
Volume 71 Number 5
Coxsackie virus B 4 valvulitis in cynomolgus monkeys
681
Fig. 3. Photomicrograph of the mitral valve lesion shown in Fig. 1, displaying round cell infiltration, fibroblastic proliferation, and stromal edema.
Fig. 4. Photomicrograph of the mitral valve lesion shown in Fig. 2, displaying round cell infiltration, stromal edema, and thickening of the chordae tendineae.
682
DePasquale, Burch, 5'un, Hale, and MogalJf!.ah
Fig. 5. Photomicrograph of a n,rrUCOllS Jesioll of th,' Coxsackie virus 13 4 74 days before autopsy.
;Iorl
i,' yalve
01 ;1
nilollioigll,
1l101l[;:")'
illonJiated with
Fig. 6. Localization of Coxsackie virus 13, antigen in the mitral valve by direct imllluuofluorcscence (X320).
Volume 71 Number 5
Coxsackie virus B 4 valvulitis in cynomolgus monkeys
also possible that other viruses in addition to Coxsackie virus may produce chronic valvular lesions in man. Inasmuch as many viruses are known to produce pericarditis and myocarditis in man, there is no basis for assuming that the endocardium is resistant to viral infection. In the present studies, viral antigen was identified in the tissues by means of direct immunofluorescent techniques. This finding, of course, suggests direct invasion of the endocardium by the Coxsackie virus. Nevertheless, some viruses, and even Coxsackie virus, may produce endocarditis through toxicity or through an autoimmune mechamsm.
4.
5. 6. 7.
8.
9.
Summary
Valvular lesions were found in 6 of 7 cynomolgus monkeys inoculated intravenously with Coxsackie virus B 4 • Typical valvular lesions of mitral stenosis were found in 2 monkeys, verrucous aortic valvulitis was found in 2 monkeys, and verrucous mitral valvulitis was found in 3 monkeys. Viral antigen was identified in the valves of the 3 monkeys in whom fluorescent antibody staining was carried out. Valvular lesions were not found in 2 monkeys inoculated with virus-free monkey kidney culture fluid. These studies demonstrate that Coxsackie virus produces valvular lesions in cynomolgus monkeys. Since Coxsackie viruses are among the most common infective agents of man, and since a substantial number of patients with chronic valvular disease give no history of rheumatic fever, it is suggested that some instances of valvulitis in man may be due to viral rather than rheumatic disease. REFERENCES 1. Lyon, E.: Virus diseases and the cardiovascular system. A survey, New York, 1956, Grune & Stratton, Inc. 2. Burch, G. E., and DePasquale, N. P.: Viral myocarditis. Ciba Foundation Symposium on Cardiomyopathies, Summit, N. J., Ciba Pharmaceutical Products, Inc., 1964, p. 376. 3. Delaney, T. B., and Fukunaga, H.: Myocarditis in a newborn infant with encephalo-
10.
11. 12. 13.
14. 15. 16.
17. 18.
19. 20.
683
meningitis due to Coxsackie virus group B, Type 5, New England J. Med. 295:234, 1958. Null, F. C., and Castle, C. H.: Adult pericarditis and myocarditis due to Coxsackie virus group B, type 5, New England J. Med. 261 :937, 1959. Sanders, V.: Viral myocarditis, AM. HEART J. 66:707, 1963. Burch, G. E., and DePasquale, N. P.: Viral endocarditis, AM. HEART J. 67 :721, 1964. Burch, G. E., DePasquale, N. P., Sun, S. c., Mogabgab, W. J., and Hale, A. R.: Experimental Coxsackie virus endocarditis, Science. In press. Kibrick, S., and Benirschke, K.: Severe generalized disease (encephalohepatomyocarditis) occurring in the newborn period and due to infection with Coxsackie virus, group B, Pediatrics 22:857, 1958. Mogabgab, W. J.: Viruses associated with upper respiratory illnesses in adults, Ann. Int. Med. 59:306, 1963. Cherry, W. B., Goldman, M., Carski, T. R., and Moody, M. D.: Fluorescent antibody technics in the diagnosis of communicable diseases, Washington, D. C., 1960, United States Public Health Service, Pub. 729. Lou, T., Wenner, H. A., and Kamitsuka, P. S.: Experimental infections with Coxsackie viruses, Arch. ges. Virusforsch. 10:451, 1960. Pearce, J. M.: Cardiac lesions in rabbits produced by a filtrable virus (Virus III), Arch. Path. 28:827, 1939. Kilham, L., Mason, P., and Davies, J. N. P.: Host-virus relations in encephalomyocarditis (EMC) virus infections. II. Myocarditis in mongooses, Am. J. Trop. Med. 5:655, 1956. Karsner, H. T.: The pathology of endocarditis. A summary review, J.A.M.A. 96:411, 1931. Tedeschi, C. G., and Stevenson, T. D.: Interstitial myocarditis in children, New England J. Med. 244:352, 1951. Godman, G. c., Bunting, H., and Melnick, J. L.: The histopathology of Coxsackie virus infection in mice. I. Morphologic observations with four different viral types, Am. J. Path. 28:223, 1952. Montgomery, J., Prinsloo, F. R., Kahn, M., and Kirsch, Z. G.: Myocarditis of the newborn, South African Med. J. 29:608,1955. Javett, S. N., Heymann, S., Mundel, B., Pepler, W. J., Lurie, H. I., Gear, J., Measorch, V., and Kirsch, Z.: Myocarditis in the newborn infant, J. Pediat. 48:1, 1956. Suckling, P. V., and Vogelpoel, L.: Coxsackie myocarditis of the newborn, Medical Proceedings 4:372, 1958. Babb, J. M., Stoneman, M. E. R., and Stern, H.: Myocarditis and croup caused by Coxsackie virus type B5, Arch. Dis. Child. 36:551, 1961.
A lthough it is known that many viruses ~ produce myocarditis and pericarditis in man,1-5 endocarditis is not generally considered to be a complication of viral infection. 6 Studies in this laboratory have shown that mural and valvular endocarditis can be produced consistently in mice with Coxsackie virus B 4.7 Because of the implications of these findings relative to the etiology of valvular heart disease in man the present study in which Coxsackie virus infection was induced in primates (cynomolgus monkeys) was performed. This paper describes the valvular lesions produced in cynomolgus monkeys with Coxsackie virus B 4 • Material and methods Virus stock. The Coxsackie virus B 4 used in these experiments was originally recovered in 1958, by Kibrick and Benirschke from a lO-day-old infant who died of encephalohepatomyocarditis. 8 The virus obtained as monkey kidney culture passage strain was prepared in rhesus monkey kidney culture. 9 Control fluid from virus-
free monkey kidney culture was also obtained. Virus and control fluid was stored at -65°C. Monkeys. Nine young adult cynomolgus monkeys were obtained from Asiatic Animal Imports, San Francisco, California. Inoculation of virus and collection of tissue. Seven monkeys were inoculated intravenously with 0.3 m!. of monkey kidney culture fluid containing Coxsackie virus B 4 105TCID 5o . The monkeys were killed 51 to 200 days after inoculation. Control experiments. Two monkeys were inoculated intravenously with 0.3 m!. of virus-free monkey kidney culture fluid. One monkey was killed 51 days after inoculation, and the other, 231 days after inoculation. Histologic studies. Serial sections of pericardium heart muscle, endocardium, and valves were stained with hematoxylin and eosin. Direct fluorescent antibody staining 10 was used to identify viral antigen in the tissues in 3 of the virus-infected animals and in 1 of the control animals.
From the Department of Medicine, Tulane University School of Medicine, and Charity Hospital of Louisiana, New Orleans, La. Supported by grants from the United States Public Health Service. Received for publication Jan. 11, 1966. *Address: Department of Medicine, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans. La., 70112.
678
Volume 71 Number 5
Coxsackie virus B 4 valvulitis in cynomolgus monkeys
Results Valvulitis was found in 6 of the 7 monkeys inoculated with Coxsackie virus B 4 and in neither of the 2 control animals (Table I). Grossly, the lesions consisted of (1) verrucous aortic valvulitis in 2 monkeys, (2) verrucous mitral valvulitis in 3 monkeys (Fig. 1), and (3) cicatricial thickening of the mitral valve leaflets and chordae tendineae with commissural adhesions (stenosis) in 2 monkeys (Fig. 2).
Table 1. Valve lesions in cynomolgus monkeys inoculated with Coxsackie virus B 4 Animal number*
Days infected
1. 5.
51 51 74
6. 7.
178 185
8. 9.
200
2.
199
Valve lesion Verrucous lesion of mitral valve Verrucous lesion of aortic valve Verrucous lesions of aortic and mitral valve Verrucous lesion of mitral valve Thick, scarred mitral valve with commissural adhesions None Thick, scarred mitral valve with commissural adhesions
*N 0 valve lesions were found in control cynomolgus monkeys Nos. 3 and 4.
679
Histologically, the valve tissue from the infected animals displayed stromal edema, round cell infiltration, fibrocytic proliferation, increased basophilia, and swelling of endothelial cells (Figs. 3, 4, and 5). In the 3 virus-inoculated animals in which direct fluorescent antibody staining was carried out, viral antigen was identified in the tissues (Fig. 6), whereas fluorescent antibody staining was negative in the single control animal in which this technique was employed. Attempts to recover Co-xsackie virus from the tissues of the infected animals were unsuccessful.
Discussion Lou, Wenner and Kamitsuka,ll using the same strain of Coxsackie virus as that employed in the present studies, found mural endocarditis in 4, and mitral valvulitis in 2, of 9 cynomolgus monkeys inoculated with the virus. The valvular lesions described by Lou and associates consisted primarily of infiltration with polymorphonuclear neutrophils and swelling of the endothelial lining cells. Although fibrocytic proliferation and commissural adhesions were not described, these workers killed the monkeys within 28 days of
Fig. 1. Photograph showing verrucous lesion of the mitral valve associated with thickening of the valve in a cynomolgus monkey inoculated with Coxsackie virus B 4 178 days before autopsy.
680
DePasquale, Burch, Sun, Hale, and
JJo.~abgab
~Jm> /-feart .! ;HO)',l f}(j/',
Fig. 2. Photograph showing tlbrosis and thickening of the mitral valve associated with commissural adhesions (stenosis) and thickening and shortening of the chordae tendineae in a cyllolllolgns monkE'v inoculated with Coxsackie virus B, 200 days before autopsy.
inoculation. Thus, the valvular lesions observed by Lou and associates were more acute than those described in the present report. It is of particular interest that valvular lesions similar to those of human mitral stenosis with commissural adhesion and thickening and shortening of the chordae tendineae and papillary muscles were observed (Fig. 2) in 2 animals in our series. In spite of the fact that attempts to recover virus from the tissues were unsuccessful, viral antigen was identified in the valves for as long as 200 days after inoculation (Fig. 6). In addition to the present studies and those of Lou and associates, viral endocarditis has been produced experimentally in mammals by Pearce,12 using Virus III in rabbits, and by Kilham, Mason and Davies,13 using encephalomyocarditis virus in mongooses. In addition, Karsner 14 and Tedeschi and Stevenson 15 have described instances of valvulitis in man which were probably viral in etiology. The present studies, as well as those of Lou, Wenner and Kamitsuka, demonstrate that valvulitis can be produced in cynomolgus monkeys with Coxsackie virus B 4 • Since Coxsackie viruses are among the
most common infective agents of man,16 it would be important to know whether these viruses produce valvular lesions in man similar to those produced in monkeys. Valvular lesions were not described in pathologic material from several epidemics of Coxsackie virus myocarditis among newborn infantsY-19 However, in none of the infants who died during these epidemics were histologic studies of the valves described. Since death usually occurred within a week to 10 days of birth, sufficient time may not have elapsed to produce gross valvular lesions. In adults, Coxsackie virus infection of the heart is not usually fatal, so that the opportunity to study pathologic material is limited. However, murmurs have been described which developed during acute Coxsackie virus myocarditis and persisted long after the infection had subsided. 20 It is well known that many patients with aortic and/or mitral valvular disease give no history of rheumatic fever. Nevertheless, the clinician is usually satisfied to render a diagnosis of rheumatic valvulitis if no other cause of the valvular lesion can be found. We think that some instances of chronic valvular disease may be due to Coxsackie virus infection. It is
Volume 71 Number 5
Coxsackie virus B 4 valvulitis in cynomolgus monkeys
681
Fig. 3. Photomicrograph of the mitral valve lesion shown in Fig. 1, displaying round cell infiltration, fibroblastic proliferation, and stromal edema.
Fig. 4. Photomicrograph of the mitral valve lesion shown in Fig. 2, displaying round cell infiltration, stromal edema, and thickening of the chordae tendineae.
682
DePasquale, Burch, 5'un, Hale, and MogalJf!.ah
Fig. 5. Photomicrograph of a n,rrUCOllS Jesioll of th,' Coxsackie virus 13 4 74 days before autopsy.
;Iorl
i,' yalve
01 ;1
nilollioigll,
1l101l[;:")'
illonJiated with
Fig. 6. Localization of Coxsackie virus 13, antigen in the mitral valve by direct imllluuofluorcscence (X320).
Volume 71 Number 5
Coxsackie virus B 4 valvulitis in cynomolgus monkeys
also possible that other viruses in addition to Coxsackie virus may produce chronic valvular lesions in man. Inasmuch as many viruses are known to produce pericarditis and myocarditis in man, there is no basis for assuming that the endocardium is resistant to viral infection. In the present studies, viral antigen was identified in the tissues by means of direct immunofluorescent techniques. This finding, of course, suggests direct invasion of the endocardium by the Coxsackie virus. Nevertheless, some viruses, and even Coxsackie virus, may produce endocarditis through toxicity or through an autoimmune mechamsm.
4.
5. 6. 7.
8.
9.
Summary
Valvular lesions were found in 6 of 7 cynomolgus monkeys inoculated intravenously with Coxsackie virus B 4 • Typical valvular lesions of mitral stenosis were found in 2 monkeys, verrucous aortic valvulitis was found in 2 monkeys, and verrucous mitral valvulitis was found in 3 monkeys. Viral antigen was identified in the valves of the 3 monkeys in whom fluorescent antibody staining was carried out. Valvular lesions were not found in 2 monkeys inoculated with virus-free monkey kidney culture fluid. These studies demonstrate that Coxsackie virus produces valvular lesions in cynomolgus monkeys. Since Coxsackie viruses are among the most common infective agents of man, and since a substantial number of patients with chronic valvular disease give no history of rheumatic fever, it is suggested that some instances of valvulitis in man may be due to viral rather than rheumatic disease. REFERENCES 1. Lyon, E.: Virus diseases and the cardiovascular system. A survey, New York, 1956, Grune & Stratton, Inc. 2. Burch, G. E., and DePasquale, N. P.: Viral myocarditis. Ciba Foundation Symposium on Cardiomyopathies, Summit, N. J., Ciba Pharmaceutical Products, Inc., 1964, p. 376. 3. Delaney, T. B., and Fukunaga, H.: Myocarditis in a newborn infant with encephalo-
10.
11. 12. 13.
14. 15. 16.
17. 18.
19. 20.
683
meningitis due to Coxsackie virus group B, Type 5, New England J. Med. 295:234, 1958. Null, F. C., and Castle, C. H.: Adult pericarditis and myocarditis due to Coxsackie virus group B, type 5, New England J. Med. 261 :937, 1959. Sanders, V.: Viral myocarditis, AM. HEART J. 66:707, 1963. Burch, G. E., and DePasquale, N. P.: Viral endocarditis, AM. HEART J. 67 :721, 1964. Burch, G. E., DePasquale, N. P., Sun, S. c., Mogabgab, W. J., and Hale, A. R.: Experimental Coxsackie virus endocarditis, Science. In press. Kibrick, S., and Benirschke, K.: Severe generalized disease (encephalohepatomyocarditis) occurring in the newborn period and due to infection with Coxsackie virus, group B, Pediatrics 22:857, 1958. Mogabgab, W. J.: Viruses associated with upper respiratory illnesses in adults, Ann. Int. Med. 59:306, 1963. Cherry, W. B., Goldman, M., Carski, T. R., and Moody, M. D.: Fluorescent antibody technics in the diagnosis of communicable diseases, Washington, D. C., 1960, United States Public Health Service, Pub. 729. Lou, T., Wenner, H. A., and Kamitsuka, P. S.: Experimental infections with Coxsackie viruses, Arch. ges. Virusforsch. 10:451, 1960. Pearce, J. M.: Cardiac lesions in rabbits produced by a filtrable virus (Virus III), Arch. Path. 28:827, 1939. Kilham, L., Mason, P., and Davies, J. N. P.: Host-virus relations in encephalomyocarditis (EMC) virus infections. II. Myocarditis in mongooses, Am. J. Trop. Med. 5:655, 1956. Karsner, H. T.: The pathology of endocarditis. A summary review, J.A.M.A. 96:411, 1931. Tedeschi, C. G., and Stevenson, T. D.: Interstitial myocarditis in children, New England J. Med. 244:352, 1951. Godman, G. c., Bunting, H., and Melnick, J. L.: The histopathology of Coxsackie virus infection in mice. I. Morphologic observations with four different viral types, Am. J. Path. 28:223, 1952. Montgomery, J., Prinsloo, F. R., Kahn, M., and Kirsch, Z. G.: Myocarditis of the newborn, South African Med. J. 29:608,1955. Javett, S. N., Heymann, S., Mundel, B., Pepler, W. J., Lurie, H. I., Gear, J., Measorch, V., and Kirsch, Z.: Myocarditis in the newborn infant, J. Pediat. 48:1, 1956. Suckling, P. V., and Vogelpoel, L.: Coxsackie myocarditis of the newborn, Medical Proceedings 4:372, 1958. Babb, J. M., Stoneman, M. E. R., and Stern, H.: Myocarditis and croup caused by Coxsackie virus type B5, Arch. Dis. Child. 36:551, 1961.