Ac~a ncuropath. (Berl.) 34, 33--40 (1976)

Acta Neuropathologica :9 by Springer-Verlag 1976

Mononucleosis-Associated Subacute Sclerosing Panencephalitis* * * F r e d H. Hoehberg, J a m e s I~. Lehrieh, a n d E. P. Richardson, Jr. Charles S. Kubik Laboratory for Neuropathology of the James Homer Wright Pathology Laboratories and the Department of Neurology, Massachusetts General Hospital and Harvard Medical School, 28oston, Mass. Paul Feorino The Viral Ontology Section, Center for Disease Control Atlanta, Ga. and the Eunice Shriver K a r l E. _~str6m Center at the Walter E. FernMd State School, Waltham, iVIass.

Summary. A thirteen-year-old girl died of subacute sclerosing panencephalitis (SSPE) which occurred as part of a complex encephalitic illness related to acute infectious mononucleosis. The cerebrospinal fluid (CSF) Epstein-Barr virus (EBV) fluorescent antibody (FA) titer was 1:64. Electron microscopic examination revealed 17 nanometer (nm) diameter paramyxovirus-like nucleocapsids in brain sections and 90 nm diameter herpes virus-like enveloped particles in negatively stained brain tissue extracts. Indirect FA staining of cerebral cortex sections demonstrated both measles and EBV antigenic material. EBV antigenic material has not previously been demonstrated in brain tissue. The proportion of B lymphocytes among the patient's peripheral blood lymphocytes was significantly increased as compared to normal controls, while the T lymphocyte percentage was normal. It is suggested that defects in cellular immunity associated with infectious mononucleosis may have been responsible for activation of latent measles-like virus. This is the tenth reported case in which two viruses have been associated with SSPE. This is the third instance in the authors' experience in whichacute EBV infection has occm'red coincident with the development of SSPE. Key words: Subacute sclerosing panencephalities -- Infectious mononucleosis -- EpsteinBerr virus -- Defective immunity.

Introduction I t is generally agreed t h a t a measles-like p a r a m y x o v i r u s plays a m a j o r role in s u b a e u t e selerosing p a n e n c e p h a l i t i s (SSPE) (tar Meulen, 1973). Measles a n t i g e n is r e a d i l y d e m o n s t r a t e d in S S P E b r a i n tissue b y i m m u n o f l u o r e s e e n t (IF) ~echniqnes, p a r a m y x o v i r u s - l i k e particles can be visualized u n d e r the electron microscope, a n d a measles-like virus has been successfully isolated from b r a i n cell cultures in several laboratories. The exact role of this measles-like virus i n the pathogenesis of S S P E is unclear, because more t h a n one virus has been i m p l i c a t e d i n a n u m b e r * Supported in part by NINDS Special Fellowship NSO 1674 (F.YI.H,), NIH grants NS0 9675 (J.R.L.) and HDO 4147 (K.E.A.) and training grant NSO 5393 (E.P.R.). ** Presented in part at the 50th Annual Meeting of ~he American Association of Neuropathologists, Boston, Mass., June 1974. 3 Aetaneuropa~h.(Berl.) Bd. 34

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o f cases ( K o p r o w s k i et al. ; S h e r m a n et al. ; U l r i c h a n d K i d d ; B e r r y et al. ; B a r b a n t i B r o d a n o et al. ; D a y a n ; J o n c a s et al., 1974b). W e r e p o r t a case o f S S P E w h i c h occ u r r e d as p a r t o f a c o m p l e x e n c e p h a l i t i c illness t o i n f e c t i o u s m o n o n u c l e o s i s , a n d in w h i c h serologic, i m m u n o h i s t o l o g i c a n d e l e c t r o n m i c r o s c o p i c evidel~ce p o i n t to t h e joint presence of a measles-like and a herpes-like virus.

Case Report A clinical and pathological description has been previously reported (Rosman et al.). A thirteen-year-old girl became apathetic, behaved inappropriately, and developed an unsteady gait. A sister reportedly had infectious mononucleosis 3 months before and had recovered without sequelae. The patient had uncomplicated measles in early childhood. On admission, the white blood cell (WBC) count was 6500 with 90/0 atypical lymphocytes. Glutamic oxaloacetic transaminase was 9 U/ml. The heterophile antibody titer was 1 : 3584 before absorption, 1:28 followiIlg beef red blood cell (RBC) absorption, and 1:1792 after guinea pig kidney antigen absorption. An ox I~BC hemolysin titer was 1 : 5000. These serological findings are consistent with a diagnosis of acute infectious mononucleosis. Cerebrospinal fluid (CSF) was colorless, under a pressure of 180 mm I-I20 and contained 5 I~BC and 8 WBC per mm a (1 polymorphonuclear neutrophil, 5 lymphocytes, and 2 monocytes), a protein of 80 mg~ and a glucose of 67 rag~ . An electroencephalogram showed generalized slowing, notably in the left frontal and temporal leads. On the second hospital day, the patient became enuretic. Her state of consciousness varied between agitation and lethargy. She complained in a dysarthrie fashion of olfactory hallucinations and made frequent paraphasic and perseverative errors of speech. There was a mild right lower facial weakness and a left extensor plantar response. Her CSF at that time contained 20 Iymphocytes per mm 3 and 125 rag~ of protein. During the next 2 days, she became less responsive and more dysphasic. By the tenth hospital day, she was unresponsive to verbal stimuli but looked about the room and moved all extremities. She was treated with dexamethasone but remained intermittently febrile, became more obtundcd and developed a right hemiparesis and myoclonus of arms and hands. On the seventeenth hospital day, CSF pressure was 210 mm H20 and there were 8 mononuclear cells per mm a, 86 mg~ of glucose and 58 mg~ of protein, 51.2~ of which was gamma globulin. Epstein-Barr virus (EBV) fluorescent antibody (FA) titers were 1:10 in CSF and 1:200 in serum. Measles complement fixation (CF) antibody titers were 1:64 in CSF and in serum. Serum CF antibody levels to herpes simplex virus were < 1:32 on day 2, and < 1:16 on day 17, to Eastern equine encephalomyelitis virus 1 : 64 on day 2 and < 1 : 16 on day 17, and to Western equine encephalomyelitis virus < 1:64 on day 2 and < 1:32 on day 17. During the next 3 weeks, the patient's clinical state improved. Spontaneous limb movement returned, but speech was limited. On the 52nd hospital day, she developed projectile vomiting, a temperature of 39.9~ C and localized seizure activity in the left arm and hand. She became unresponsive with fixed and dilated pupils, and died on the 57th hospital day.

Methods Post-Mortem Examination. The post-mortem examination was performed 41/2 hrs after death. Portions of frontal, parietal, and occipital cortex, cerebellum and medulla were removed under sterile conditions and prepared for further study as noted below. The remainder of the brain was fixed in 10~ formalin for 14 days and sectioned coronally in I cm thick sections. Paraffin and eelloidin embedded sections were stained using the Loyez method for myelin and with hematoxylin-eosin and eresyl violet. Electron Microscopy. Sections of formalin fixed cortex and white matter were posVfixed in osmium tetroxide and embedded in Epon (Shell Oil Co.). Cortical "extracts" were prepared according to a procedure designed to concentrate and purify herpes virus particles present in brain tissue; this method will subsequently be described in detail by one of us (P.F.). Samples of cortex and white matter, flash frozen on dry ice and

Mononucleosis-Associated SSPE

35

stored at --70~ were ground and sonicated and then purified by eentrifugation on a 300/0 sucrose solution followed by concentration and separation using a series of potassium tartrate and glycerol gradients. A portion of this material was filtered and negatively stained with uranyl acetate.

Primary Explant Cultures. Tissue samples from frontal and parietal cortex were obtained under sterile conditions and cells cultured using methods previously reported (Katz et al.). Immunofluorescence (IF). Sterile samples of frontal and parietal cortex were flash frozen on dry ice and stored at --70~ Five mieron thick cryostat sections of these samples were fixed in acetone and stained for measles and EBV antigens in separate indirect FA systems (ter Meulen et al., 1970). Explant tissue culture cells which had been co-cultivated with African green monkey kidney cells (CV-I strain) were grown on eoverslips, fixed in acetone and stained for both antigens in a similar fashion. Sections of cerebrum from a child who had died as a result of cerebrovascular accident, and CV-I cells infected with the Woodfolk strain of measles virus, served as controls. A. Measles Virus. Tissues were overlaid with rhesus monkey anti-measles serum (CF titer 1:128) and eounterstained with fluorescein isothiocyanate (FITC) --conjugated rabbit anti-monkey IgG. The antisera were obtained commercially (Microbiological Associates, Bethesda, Md.). B. Epstein-Barr Virus. Tissues were overlaid with human serum with an EBV FA titer of 1:400 from which measles F A antibodies had been adsorbed with measles antigen and counterstained with FITC-eonjugated rabbit anti-human IgG (Microbiological Associates). Measles F A antibodies were adsorbed by mixing EBV antiserum and measles hemagglntinating antigen (Microbiological Associates) at a 10:1 ratio, incubating 3 hrs at 23~ and 12 hrs at 4~ and then centrifuging (2000 rpm) 1 hr at 4~ Control tissue sections were overlaid only with fluoreseein-conjugated rabbit anti-human IgG. T and B Cells. A sample of venous blood obtained on the 28th hospital day was evaluated to determine relative populations of T and B cells. T cells were identified by spontaneous formation of rosettes in a sheep RBC suspension (Oger et al.). B cells were identified by rosettes formed with human complenent-aetivated sheep RBC. Results General autopsy was unremarkable except for atypical large cells with intensely basophilic convoluted nuclei and irregular cellular outlines found in the spleen, lungs, liver, and lymph nodes. The lymph nodes showed preserved architecture with plump histJoeytes interspersed with Iymphocytes. These are not the changes of acute infectious mononucleosis; the patient died several weeks after that diagnosis was first made, however. The brain weighed 1250 g and was swollen. On microscopic examination there was a diffuse encephalomyelitis involving cerebral cortex, diencephalon, brainstem and cervical spinal cord. Sections stained for myelin showed large areas of destruction of myelinated fibers. The cerebral cortex, and to a lesser extent the basal ganglia and thalamus, showed an extreme degree of neuronal loss. These regions appeared hypercellular, however, because of the presence of large numbers of reactive histioeytes, some grouped into focal aggregates (Fig. 1). Astroeyrie proliferation was evident, but there was little or no fibrillary gliosis. In all parts of the brain, there were prominent perivascular infiltrates consisting mainly of lymphoeytes and larger mononuctear cells, without evidence of atypical cells (Fig.2). Rare Cowdry type A inclusions were seen within neuronal and oligodendroglial nuclei (Fig. 3). Electron Microscopy. I n c l u s i o n b o d i e s c o m p o s e d o f t u b u l a r f i l a m e n t s w i t h a n o u t e r d i a m e t e r o f a p p r o x i m a t e l y 17 n m w e r e f o u n d w i t h i n o l i g o d e n d r o g l i a l n u c l e i (Fig. 4). T h e s e f i l a m e n t s w e r e i d e n t i c a l in a p p e a r a n c e to t h e p a r a m y x o v i r u s - l i k e n u c l e o c a p s i d s s e e n in n e r v e a n d glial cells in o t h e r eases o f S S P E . N o o t h e r abn o r m a l s t r u c t u r e s w e r e s e e n in t h e b r a i n s e c t i o n s u n d e r t h e e l e c t r o n m i c r o s c o p e . Examination of the negatively stained purified brain extracts disclosed spherical 3*

36

Figs. 1--8

F. It. Hochberg et al.

Mononucleosis-Associated SSPE

37

structures with capsids 90 nm in diameter surrounded by 110 nm envelopes, an appearance suggestive of herpes virus particles (Fig. 5). Similarly processed brain extracts from two individuals who did not have SSPE, did not show herpes-like particles. Primary Explant Cultures. Primary cultures of brain cells were established but did not survive attempts at subeultivation. Primary brain cells were co-cultivated with cells of the CV-1 strain of African green monkey kidney, and the resulting cells subcultured for 20 serial passages. No eytopathie effects were detected in the primary brain cell culture or in the co-cultivated cell line. The co-cultivated cells were negative for measles and EBV antigens when stained by the indirect FA method. Immuno/luorescence o] Brain Tissue Sections. Sections of frontal and parietal cortex stained for measles antigen showed bright diffuse cytoplasmic and dense nuclear fluorescence in neurons and astrocytes (Fig.6). Brightly fluorescent multiple inclusion bodies were seen in several cells (Fig.7). Sections of fror=tal cortex, stained with anti-EBV serum, showed diffuse and speckled cytoplasmic fluorescence in neurons (Fig. 8) and in perivascular cells (presumably lymphocytes), but not in astrocytes. Sections stained only with fluorescein-conjugated rabbit anti-human IgG showed no fluorescence. Sections of brain from a child who had died of a cerebrovaseular accident showed neither measles nor EBV antigen. I t was found that all available high-titer human antisera to EBV contained FA to measles virus as well, as determined by indirect I F of measles-infected CV-1 cells. Removal of measles FA antibodies from these EBV antisera by adsorption (see Methods) eliminated F A staining of the measles-infected CV-1 cells, but did not change the neuronal fluorescence demonstrated by I F staining of cortical sections with EBV antisera. T and B Cells. Spontaneous rosette forming cells (T cells) represented 51 ~ of the patient's lymphocytes. Control patients evaluated by this technique, have 56 :k 30/0 (SEM) of their lymphocytes as T cells (0get et al.). Activated human

Fig. 1. Cerebral cortex showing focus of histiocytes--a microglial nodule Fig.2. Cortical white matter (• vascular reaction

with prominent lymphocytic and mononuclear peri-

Fig. 3. Cortical neuron with large intranuclear inclusion Fig.4. Electron micrograph showing 17 nm diameter tubular filaments present within inclusion body of oligodendroglial nucleus Fig.5. Electron micrograph of negatively stained purified brain material showing spherical structure 90 nm in diameter with 110 nm envelopes Fig. 6. Dense nuclear and diffuse cytoplasmic fluorescence of corticM neurons stained indirectly with hyperimmune measles antiserum :Fig.7. Densely fluorescent inclusion bodies within a cortical cell stained indirectly with hyperimmune measles antiserum Fig. 8. Diffuse and speckled neuronal cytoplasmic fluorescence in cortical sections stained indirectly with hyperimmune E-B virus antiserum

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F.H. Hochberg et al.

complement receptor cells (B cells) made up 46 ~ of the patient's lymphocytes. In control patients evaluated by this technique, 18 ~= 2~ (SEM) of lymphocytes were B cells (Oger et al.). Discussion

The case reported represents 1 of 3 similar cases of SSPE coincident with acute EBV infection. A detailed examination of all 3 cases will be published separately. Two distinct conditions, SSPE and infectious mononucleosis, were diagnosed in this case. The diagnosis of SSPE was based on pathologic findings of a chronic diffuse encephalitis with Cowdry type A inclusion bodies in a patient who died during a subaeutely evolving myoclonic encephalopathy. The elevated levels of measles antibody and gamma globulin in CSF supported the diagnosis. The diagnosis of infectious mononucleosis was based on the abnormal heterophile agglutinin, ox cell hemolysin and EBV antibody titers. The evidence also points to the presence of two different viruses in the brain of the patient, a measles-like paramyxovirus and an EBV-like herpes virus. The presence of measles-like virus was indicated by the elevated titers of measles antibodies in CSF, by I F demonstration of measles antigen in neuron and astrocytes and by the electron microscopic findings of paramyxovirus-]ike nucleocapsids in oligodendroglial nuclei. The presence of EBV in brain was suggested by EBV antibody present in CSF, by the herpes-like particles seen on electron microscopic examination of purified brain extracts and by the I F demonstration of EBV antigen in neurons. Although it is possible that the EBV antibodies in this patient's CSF had diffused passively from her serum, in our experience it is unusual to find any EBV antibody in CSF. No EBV antibody was present in 40 CSF samples from patients with infectious mononucleosis studied by one of us (P.F.). One ease of mononucleosis with elevated CSF EBV antibody levels has been reported, however (Joncas et al., 1974a). There have been 9 cases previously reported in which a pathologic diagnosis of SSPE was made and a virus other than measles implicated (Sherman et al. ; Ulrich and Kidd; Berry et al.; Barbanti-Brodano et al.; Dayan; Joncas et al., 1974b). In 5 of the 9 cases, a herpes-like virus was suggested (Shermann et al. ; Ulrich and Kidd; Berry et al. ; Dayan; Joncas et al., 1974b). In only 1 of these 5 cases (Dayan) was a herpes virus (herpes simplex) successfully isolated ; and in none was a measles-like virus isolated. In 2 of the putative herpes virus-associated SSPE cases (Berry et al. ; Dayan), both paramyxovirus-like nuc]eocapsids and herpes-like particles were seen under the electron microscope in brain tissue, but these cases differ from the one reported here in showing extensive necrosis of a type usually associated with herpes simplex encephalitis. In 3 of the remaining 4 cases, papova-like virus particles were seen under the electron microscope in cell cultures derived from brain tissue (Barbanti-Brodano et al.); a measles-like virus was isolated from two of these cases, but no other viral agents could be recovered. In the last ease (Joncas et al., 1974b), adenovirus type 21 was identified in a co-culture of kidney cells obtained at autopsy and Itep-2 cells (human laryngeal carcinoma).

Mononucleosis-Associated SSPE

39

Other workers have presented epidemiological evidence to suggest a role for 2 viruses in SSPE (Joncas et al., 1974b; Brody el al. ; Baguley and Glasgow). They have noted a history of previous coincident measles and chicken pox infections among a proportion of SSPE patients (Brody et al.), a possible increase in the incidence of SSPE following the introduction of the Salk poliomyelitis vaccine (Baguley and Glasgow) and, most interestingly, the finding of elevated EBV antibody levels in sera and CSF from SSPE patients (Joneas et al., 1974b). The role of infectious mononucleosis and EBV or other herpes-like virus in the pathogenesis of our patient's SSPE is uncertain. The EBV might be unrelated to SSPE, a commensal found by chance. The patient may have been predisposed to both SSPE and infectious mononucleosis by an underlying immunological defect. The herpes-like virus might be the etiologic agent for SSPE, the measles-like virus acting as a syncytia-forming agent to allow the spread of the herpes-like virus ; a concept first proposed by Koprowski el al. to relate papova-like and measles-like viruses in SSPE. The explanation most attractive to us relates a mononucleosis-associated immunosuppression to the pathogenesis of SSPE in this case and possibly in others as well. The patient's failure to show the expected (Sheldon etal. ; Pattengale et al.) increased proportion of peripheral blood T lymphocytes in response to her infectious mononucleosis might be associated with defective cell-mediated immunity. An apparent lack of T cell proliferation has been noted previously in fatal infections mononucleosis (Bar et al.). Additional defects in cellular immunity have been found associated with infectious mononucleosis as well (Sheldon et al. ; Bar et al. ; Lanthorp et al. ; Mangi et al.). We suggest that mononucleosis-associated changes in cellular immunity might allow activation of latent SSPE paramyxovirus, in a fashion analogous to the role suggested for exogenous immunosuppression in studies of both experimental (Wear and Rapp) and clinical SSPE (Breitfield et at. ; Sluga et al.). Acknowledgement. The authors wish to thank Dr. Joel Oger for performing the T and B cell assays; Dr. W. ttenle for his valuable advice and assistance; Dr. B. G. W. Arnason for his comments on the manuscript; and Mr. Arnold Freedman and Miss Claire Liu for their valuable technical assistance.

References Baguley, D. M., Glasgow, G. L. : Subacute sclerosing panencephalitis and Salk vaccine. Lancet 1973 II, 763--765 Bar, 1~. S., DeLor, C.J., Clausen, K. P., Hurtubise, P., Henle, W., ttewetson, J. F.: Fatal infectious mononucleosis in a family. New. Engl. J. Med. 290, 363--367 (1974) Barbanti-Brodano, G., Oyanagi, S., Katz, lVI.,Koprowski, H. : Presence of two different viral agents in brain cells of patients with subacute sclerosing panenccphalitis. Proc. Soc. exp. Med. 134, 130--246 (1970) Berry, R. G., Chambers, R. A., Cracco, J. : SSPE associated with two viruses. VIe International Congress of Neuropathology. Paris, Aug. 31--Sept. 4, 1970, pp. 1019--1020 Breitfield, V., Hashida, Y., Sherman, F. E., Odagiri, K., Yunis, E. J. : Fatal measles infection in children with leukemia. Lab. Invest. 28, 279--290 (1973) Brody, J. A., Detels, R., Sever, J. L. : Measles antibody titers in sibships of patients with subacute sclerosing panencephalitis and controls. Lancet 1972I, 177--178 Dayan, A. D. : Encephalitis due to simultaneous infection by herpes simplex and measles virus. J. neurol. Sci. 14, 315--323 (1971)

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Joneas, J. H., Chicoine, L., Thivierge, R., Bertrand, M. : Epstein-Barr virus antibodies in the cerebrospinal fluid. Amer. J. Dis. Child. 127, 282--285 (1974a) Joncas, J., Geoffory, G., McLaughlin, B., Albert, G., LaPointe, N., David, P., LaFontaine, P~., Granger-Julien, M.: Subacute sclerosing encephalitis. J. neurol. Sci. 21, 381--390 (1974b) Katz, M., 0yanagi, S., Koprowski, H.: Subacute sclerosing panencephalitis. Structures resembling myxovirus nueleocapsids in cell cultured from basis. Nature (Lond.) 222, 888--890 (1969) Koprowski, H., Barbanti-Brodano, G., Katz, M. : Interaction between papowa-like virus and paramyxovirus in human brain cells: a hypothesis. Nature (Loud.) 224, 1054--1047 (1970) Lanthorp, K., Wahren, B., Hanngren, A. : Infectious mononucleosis and depression of cellular immunity. Brit. J. Mcd. 1972II, 668--669 Mangi, R. J., Neiderman, J. C., Ke]leher, J. E., Jr., Dwyer, J. M., Evans, A. S., Kantor, F. S.: Depression of cell-mediated immunity during acute infectious mononucleosis. New Engl. J. Med. 291, 1149--1153 (1974) Oger, J., Arnason, B. G. W., Wray, S. It., Kistler, J. P. : A study of T and B cells in multiple sclerosis. Neurology (Minneap.) 25, 444 (1975) Meulen, V. ter, Katz, M., Muller, D.: Subacute sclerosing paneneephalitis: a review. Curr. Trends Microbiol. Immunol. 57, 1--38 (1973) Meulen, V. ter, MiilIer, D., Katz, M., K~iekell, M. Y., Pattengale, R. K., Smith, R. W., Perlin, E.: Atypical lymphocytes in acute infectious mononucleosis. New Engl. J. Med. 291, 1145--1148 (1974) 1%osman, P., ttochberg, F., Richardson, E. P., Jr. : Dementia rapidly progressing to coma in a thirteen-year-old girl. Case records of the Massachusetts General Hospital. New Engl. J. Med. 29, 141--149 (1974) Sheldon, 1%.J., Hensted, E. H., Papamichail, M., ]-Iolborow, E. J. : Thymie origin of atypical lymphoid cells in infectious mononucleosis. Lancet 1973 I, 1153--1555 Sherman, F. E., Davis, R. L., Haymaker, W.: Subacute inclusion encephalitis--report of a case with observations on the fluorescent anti-herpes simplex antibody reaction. Aeta neuropath. (Berl.) 1,271--288 (1961) Slugs, E., Budka, H., Jellinger, K., Piehler, E. : SSPE-like inclusion body disorder in treated childhood leukemia. Acts neuropath. (Berl.) Suppl. VI, 267--272 (1975) Toppich, G. : Immunohistological, microscopical, and neurochemical studies on encephalitides. IV. Subacute sclerosing (progressive) panencephalitis: histochemical and immunohistological findings in tissue cultures derived from SSPE brain biopsie. Acta neuropath. (Berl.) 15, 1--10 (1970) Ulrich, J., Kidd, M. : Subacute inclusion body eneephalitis--a histological and electron microscopic study. Acts neuropath. (Berl.) 6, 359--370 (1966) Wear, D. J., Rapp, F. : Latent measles virus infection of the hamster central nervous system. J. Immunol. 107, 1593--1598 (1971) Received June 13, 1975; Accepted October 4, 1975

Dr. James 1%.Lehrich Dept. of Neurology Massachusetts General I-Iospital Fruit Street Boston, Mass. 02114 U.S.A.