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Herpesviruses in chronic encephalitis associated with intractable childhood epilepsy
Herpesviruses in Chronic Encephalitis Associated With Intractable Childhood Epilepsy HARRY V. VINTERS, MD, REBECCA WANG, AND CLAYTON A. WILEY, MD, PHD
MS,
atrophy, scarring, and chronic inflammation manifesting as microglial nodules and perivascular cuffing by mononuclear inflammatory cells, with microvascular proliferation. ‘-I ’The cortex and white matter may be affected to variable degrees.‘” The definitive establishment of a specific viral etiology for RE has proven elusive. However, over the past few years in situ hybridization has been used to show that cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV) may be found within the inflamed central nervous system (CNS) tissue,‘“-‘” although using comparable methodology these pathogens have not been found in all resected RE tissues.” Additionally, enterovirus-like particles have been found by electron microscopy in a child with the related syndrome of hemiplegia, hemiconvulsions, and epilepsy. Ix We extended the search for a specific viral etiology fat RE and chronic encephalitis associated with epilepsy using the sensitive technique of polymerase chain reaction (PCR) on resected brain tissue.
Herpesviruses (especially cytomegalovirus [CMV] and Epstein-Barr virus [EBV]) have been implicated in the pathogenesis of chronic (Rasmussen) encephalitis associated with epilepsy. To assess the presence of herpesvirus genes in brain tissue from epileptic children with chronic (usually Rasmussen type) encephalitis, DNA was extracted from surgically resected brain tissue and studied by the polymerase chain reaction using primers specific for CMV, varicella zoster virus, herpes simplex virus, EBV, and human herpesvirus 6 genes. By this technique evidence for the presence of low levels of CMV and EBV genes was detected in most brain specimens from encephalitis patients and in several brain specimens from patients without encephalitis (eg, cortical dysplasia, gliosis, or encephalomalacia) who also had intractable epilepsy. Occasionally, both EBV and CMV genes were found in the same brain. Signal strength for both CMV and EBV was much lower in epileptic brains than in the brains of acquired immunodeficiency syndrome patients with CMV encephalitis or brain lymphoma. We found evidence for infection of the brain by human herpesvirus 6 in only one patient with encephalitis. Polymetase chain reaction technology applied to resected brain tissue from epileptic patients may provide evidence for or against viral-mediated pathogenesis of Rasmussen encephalitis or other types of encephalitis. The small amounts of EBV and CMV genes found suggest that herpesvirus infection of the brain does not directly cause Rasmussen encephalitis. HUM PATHOL 24:871879. Copyright 0 1993 by W.B. Saunders Company
MATERIALS
AND METHODS
Study material
Among recognized neuropathologic substrates of intractable focal epilepsy in children is a form of chronic encephalitis characterized initially by Rasmussen and colleagues.‘.” Clinically, Rasmussen-type encephalitis (RE) presents with uncontrollable unilateral focal seizures or epilepsia partialis continua associated with a progressive neurologic deficit (usually hemiparesis sometimes advancing to hemiplegia), with variable intellectual impairment. ‘-’ Resection of the affected brain tissue may ameliorate the condition.“-“’ The resected brain tissue shows features of a chronic encephalitis with
who undetwetit
included brain tissues frown seven children I esections for epilepsy extensive focal cortical
Six patients presented with typical clinical Ceatures suggestive of RE. One child had a poorly documented history of encephalitis at the age of 2 years with subsequent onset of an intractable seizure disorder. The mean age of symptom onset in the seven patients was 48.6 months (range, 24 to 72 months), while the mean age at surgery was 10 I .4 months (range, 4 1 to 172 months), yielding a mean interval of disease onset to sul-gery of 52.8 months (range, 8 to 1 14 months). The typical histopathologic features of RI?‘,“,‘” were seen in six patients (Fig 1); the child with a questionable antecedent history of encephalitis at the age of 2 years had the additional unusual finding of numerous multinucleatecl giant c,ells within the resected tissue (Fig 1C). A detailed report on the light microscopic, ultrastructural, and inimurlohistochemical features (emphasizing macrophage, microglial, major histocompatihility complex, and lymphocyte markers) of the resected tissue is in preparation. Control tissues from which DNA was rsolated included (I) paraffin sections derived from pellets of cultured glial or fibroblast cells (either noninfected or infected with CMV, varicella roster virus [VZV], or HSV); (2) sections of pelleted Iymphoblastoid cell lines either noninfected or infected with human herpesvirus 6 (HHV-6); (3) paraffin sections of pellets of R+ji cells (American Type Culture Collection, Rockville, MD) derived from human Rurkitt lymphoma and known to contain ERV genome or non-EISV-infected lymphoid cells (a gift of Dr S. David Hudnall, Department of Pathology alid l>aboratory Medicine, I J<:I,A): (4) tissue con-
From the Department of Pathology and Laboratory Medicine, and the Brain Research Institute, UCLA Medical Center, Los Angeles. <:A; and the Department of Patholob7/, UC San Diego School of Medic,ine, La Jolla, CA. Accepted for publication January 1.5, 1993. Supported in part by Public Health Service (irants No. NS 26312 ,md 28383 (to H.V.V.) and by a grant from the University of California AIDS Task Force, Public Health Service &ants No. NS 25178, 27417, and 27810, and MH45294, and funds from the Robert H. Fer-nrr Fund (to C.A.W.). KI,~ wor&: chronic (Rasmussen type) encephalitis, epilepsy, herpesviruses, polymerase chain reaction. Address correspondence and I-eprint requests to Harry V. Vinters, MD, Department of Pathology and I.aboratory Medicine, CHS 1% 170. UCI,A Medical Center, Los Angeles, CA 90024-1732. Copyright 0 1993 by W.B. Saunders Company 0046-8 I77:r,s!2408-ooo8$5.00/0
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FIGURE 1. Histopathologic features of chronic encephalitis associated with pediatric epilepsy. Panels A and B show features characteristic of RE. (A) Resected brain tissue (patient no. 2, Table 2) shows extensive chronic inflammation centered on microvessels and prominent astrocytic gliosis. (Hematoxylin-eosin stain; original magnification x85.) (B) Brain tissue (patient no. 3, Table 2) shows transmural chronic inflammatory cells in two microvessels, with extension of the inflammatory infiltrate into brain parenchyma. The degree of tissue injury (with attendant gliosis) is much less pronounced than in the example illustrated in panel A. (Hematoxylineosin stain; original magnification X560.)
trols, including paraffin sections of autopsy brain from paCents with the acquired immunodeficiency syndrome (AIDS) and known CMV encephalitis or primary CNS lymphoma that 1~36positive for EBV; and (5) resected brain tissue from children (of approximately comparable age) with intractable seizures who had focal cortical dysplasia, heminiegalencrphaly, or other tiialformati~ejtlegenerative lesions that were lacking ;I major inflattltttatot.~ cotttpotlettt.‘g.“‘~~” The lattet control group seemed appropriate Biven that certain cerebral tnalfot.tnations (eg. pa~hygyrta-lissetl~~phaly) havr been associated with herpesvirus infections, such as COIIgenital CICIV infection.“.‘” Careful review of‘ brain sections from control gmup no. 5 revealed negligible or absent evidence for chronic inflatttmation in all cases. It should be noted, howevet-, that the children with nonencephalitic lesions underwent surgery at ;I somewhat younger age than those with encephalitis (6!) u 101.4 months). The neuropathologic change in brain tissue from this group has been described in detail.“~“‘~”
III all cases genomic DNA was isolated from paraffin sections of brain using slight tnodifications of established techniques.” For two patients witlt RE and five patients with nonintlattit~iatot-y cortical lesions, fresh-frozen tissue (P800C from the time of surgery) was available for study. Briefly, paraffin sections (20 X 10 ~111thickness) were placed in a I .5-ml. Ep},etldorftrti~rofi~ge tube and treated with Histoclear to dissolve the paraffin; this material was centrifuged and the pellet was treated with 100% ethanol, which was discarded following centrifugation. From this point the material initially derived from the tissue pellet and the freshly homogenized (in .50 mmol/I, Tris-HCI) brain wrre treated in at1 identical fashion. Tissue was digcstcd Fvith proteinase K (final concentration, 200 p(g/ml,) in a digestion buffer containing 50 mmol/l. Tris (pH 8.5), I mmol/l. cthylcnediatnine tetraaretic acid, 0.5% to 1 .O% sodiuttl dodec\;l sulfate, and 0.1 or 0.05 N NaCI at 55°C for 3 horn-s. Proteinase K was inactivated by heating the tubes for 30 minutes at 05°C:. The supernatatlt was treated with 5 mol/l 1K ac~tatc and subsequently with 7.5 mol/L NH., acetate
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(Vinters et al)
and 100% ethanol for at least 2 hours at --8O’C. ‘The DNA pellet was resuspended in Tris-ethylenediamine tetraacetic acid, pH 7.8, and OD,,, was measured in a spertrophotometer. Although DNA yields were quite variable from case to case, 250 ~1~ of a 10% wt/vol fresh brain tissue homogenate 01 20 X 10 Fin-thick paraffin sections usually yielded a final DNA concentration of 0.25 to 1 .O pg/pL. Approximately 0.5 to 1 .O pg of DNA was used as the starting material for each PCR reaction. To minimize cross-contamination DNA isolations were carried out in a room separate from that used for preparation of the final PCR incubation mixtures and all PCR specimen mixtures were prepared in a laminar tlow hood. Isolated DNA was assayed for the presence of genes specific to five different human herpesviruses using primer pairs as shown in Table 1. Isolated DNA also was simultaneously or sequentially amplified for a gene encoding glyceraldehyde 3phosphate dehydrogenase (GASPD) as a “housekeeping” gene to assess the integrity of DNA from each specimen. Polymerase chain reaction was carried out in a Perkin Elmer Cetus (Norwalk, CT) DNA thermal cycler. Each 50-PL rnixture subjected to PCR included 0.5 to 1 .O pg of template DNA from human brain or cultured cell blocks, 30 pmol of each primer pair, 2.5 mmol/L MgC&, and 200 ~~nol/L, dNTP. Thermal cycling was carried out as follows: one cycle of 94°C (5 minutes) and 65°C (2 minutes), 30 cycles of 94°C (2 minutes) and 65°C (2 minutes), and one cycle of 72°C (7 minutes). Polymrrase chain reaction products were electrophoresed on an 8% polyacrylamide gel, which was then stained with ethidium bromide and photographed with W transillumination. For CMV. EBV, and HHV-6, specific viral gene probes complementary to the amplified DNA product were end-labeled with “P adenosine triphosphate using T4 polymerase kinase and hybridized to the PCR product after denaturation of the DNA by boiling.“” Activity of the specific probe was usually between 1.5 X 10’ and 1.5 X 10” cpm/sample. The hybridization mixture was electrophoresed through an 8% polyacrylamide gel, autoradiograms of which were then obtained at brief (2- to ?-hour) or overnight exposures at -80°C.
RESULTS FIGURE 1. (Continued) (C) Tissue from patient no. 5 (Table 2) shows marked astrogliosis and perivascular inflammation and the unusual feature of occasional perivascular multinucleated cells (arrow). The latter feature was only noted in one patient and is distinctly unusual in RE. (Hematoxylin-eosin stain; original magnification X220.)
TABLE (kwe to he Atnplitied
1.
In all cases a single visible band of variable intensity was identified on ethidium bromide-stained gels of PCR products obtained with GA3PD-specific primers, indicating the presence of adequate DKA. The band always
Herpesvirus Primer Pairs Used in Polymerase
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271 194 118
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FIGURE 2. Ethidium bromide-stained polyacrylamide gels of PCR products obtained after amplification of DNA from human brain tissue. The top panel represents results of PCR using primer pairs for GA3PD gene and the lower panel represents results of PCR with primer pairs specific for CMVMIE genes. M, molecular weight standards (no. of base pairs); -, negative control, DNA from noninfected U373 cells; f, positive control, DNA from AD169 CMV-infected U373 cells. Samples included brain specimens from patients with encephalitis (E lanes), nonencephalitis (epilepsy) cortical resections (D lanes), and AIDS (autopsy specimen) with CMV encephalitis (lane A). Note the variation in intensity of the amplified GA3PD products despite loading approximately equivalent amounts of DNA in each PCR mixture. The only detectable amplified CMV product is in the control infected cells and brain tissue from the AIDSKMV encephalitis patient. All starting material for DNA extractions in this study was paraffin embedded.
was more intense using DNA derived from freshly harvested (and frozen) rather than paraffin-embedded brain tissue. DNA extracted from paraffin sections of cultured cells infected with CMV, VZV, HHV-6, or HSV, or from sections of Raji cells yielded intense bands on ethidium bromide-stained gels of PCR products at the expected molecular weight (Figs 2 to 4). Polymerase chain reaction products using template DNA obtained from epilepsyderived brain tissue and assessed by ethidium bromide failed to show the presence of CMV, HSV, VZV, HHV6, or EBV genes in the brain tissue (Figs 2 to 4). In contrast, using this method CMV genes were easily detected in brains from AIDS patients with CMV encephalitis (Fig 2). Polymerase chain reaction carried out on AIDS-related CNS lymphoma tissue using EBV primers yielded distinct bands at the expected molecular weights in over 50% of the cases (unpublished data); representative cases only are shown in Fig 4. The use of ‘2P-radiolabeled specific probes and autoradiography to detect the labeled gene significantly increased the sensitivity of detecting the PCR product. Using this technique trace or weak signals for CMV were found in at least one sample from six 874
of seven cases of encephalitis (Fig 5). The signal, however, was faint in most of these specimens even after prolonged exposure of the autoradiogram and use of an intensely labeled specific probe. In contrast, identically prepared autopsy tissue from AIDS patients with known CMV encephalitis, even of a mild degree with rare identifiable inclusions, yielded an intense band on autoradiography, essentially comparable to that seen with cell culture controls. It was of interest that brain specimens from two of six noninflammatory lesions also showed CMV, yielding a signal comparable in intensity to that noted in tissue from patients with encephalitis. Similar observations were made with EBV primers and probes. Trace or weak EBV-specific bands were noted on autoradiograms using brain tissue derived from five of seven patients with encephalitis and in two of six nonencephalitis lesions. It is noteworthy, however, that the most intense EBV-specific band was repeatedly found in the resected brain of a patient who did not show lesions of RE, but had severe hydrocephalus with secondary gliotic and destructive lesions in the cerebral cortex (Fig 6). Results from all specimens are summa-
HERPESVIRUSES
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603
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2710 194118FIGURE 3. Ethidium bromide-stained gels of PCR products (DNA extracted from paraffin sections) using primers for GA3PD (top panel) and VZV (bottom panel). The lanes represent tissue from various sources (see Fig 2). The VZV gene is detectable only in the cultured infected cells.
603271194118-
rized in Table 2. Note that several patients showed evidence of both CMV and EBV in the brain by PCR. A weak signal suggestive of the presence of HHV6 was found after liquid hybridization of DNA extracted from only one of several paraffin tissue blocks from a single patient with RE; the radioactive signal was noted only after prolonged exposure of the autoradiogram (data not illustrated).
RE.“‘.“’ In view of our previous inabilitv to detect CMV in RE brain specimens by in situ hybridization,” we felt it important to extend our observations using highly sensitive PCR methodology. Even though we have not used quantitative PCR our results suggest that low levels of EBV and CMV genes may be found in resected brain specimens that show neuropathologic features of encephalitis, as well as in some specimens from epileptic patients without encephalitis (eg, with changes of cortical dysplasia OI other types of secondary destructive lesion). Considering our negative in situ hybridization studies with CMV PCR seems to be more sensitive for detecting probe,” small amounts of viral DNA, although the purpose of the present investigation was not a direc.t comparison of the sensitivity of in situ hybridization versus PCR. Given the sensitivity of PCR for detecting small numbers of gene copies, we cannot rule out the possibility that we have amplified blood-borne viral genes in the PCR reaction; indeed, there is no way to rule this out in the present experimental paradigm.
DISCUSSION The highly sensitive technique of PCR’4,‘” can be used to detect minute quantities of numerous genes in human tissues, whether derived from autopsy or biopsy and regardless of whether the template DNA is derived from paraffin blocks or from freshly isolated tissues.“‘-*” In this initial study of viral genes within the resected brain of patients with intractable seizures secondary to chronic (usually Rasmussen type) encephalitis, we have chosen to emphasize herpesviruses because of several reports suggesting that CMV and EBV may be of pathogenetic significance in the causation of 875
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FIGURE 4. Ethidium bromide-stained gels of PCR products obtained using DNA from the surgically resected brain tissues of a 3-month-old child with severe cortical dysplasia causing epilepsy (lane D), of four patients with histologically verified encephalitis (E lanes), and of four representative patients (autopsy tissue) with CNS lymphoma (L lanes). The top panel represents the results of PCR performed using primer pairs for a GABPD gene and the bottom panel represents the results of PCR using primer pairs for the IR3 region of the EBV genome. All starting material for this study was paraffin-embedded tissue. M, molecular weight standards.
1180
603271
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194118 -
m
-+
1
872603-
271-
FIGURE 5. An autoradiogram (2-hour exposure) of gel representing electrophoresed PCR products (using CMV primer pairs) hybridized with a 32P-labeled CMV probe. The lanes are as follows: m, molecular weight standards; -, negative cell culture control; and +, positive ceil culture control. Lanes I, 2, 3, and 8 to 12 represent PCR using DNA from histologically proven encephalitis cases; lanes 4, 5, and 7 represent PCR using DNA from nonencephalitis epilepsy cortical resections; and lane 6 represents PCR using an autopsy brain specimen from an AIDS patient with CMV encephalitis (positive tissue control). Lanes 1 and 2 represent DNA extracted from two different paraffin blocks from the same patient. All lanes except lanes 3 to 5 represent DNA isolated from paraffin sections; the DNA in lanes 3 to 5 was from fresh-frozen tissue. A faint signal was detected in lanes 9 and 10. After overnight exposure, signal was noted in lanes 1 and 7 to IO.
HERPESVIRUSES
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123456789
10
11
12
872603FIGURE 6. (Top) Autoradiogram (2-hour exposure) of gel representing PCR products (using EBV probe) hybridized with a radiolabeled probe. The apparent molecular weight of hybridized product is approximately 600 base pairs. The lanes are the same as in Fig 5, except that lane 6 contains PCR product from an AIDS patient with CNS lymphoma (positive tissue control) and lanes 7 and 8 are reversed (ie, lane 7 is from a patient with encephalitis and lane 8 is from a patient with cortical dysplasia). Note the detectable bands in lanes 4 and 7 to 11. After overnight exposure (a portion of the autoradiogram is shown in the bottom panel) signal was noted in lanes I, 4, and 7 to 11, in addition to the control lanes. The most prominent signal is in lane 4, representing resected brain tissue from an epileptic patient without features of encephalitis
271-
194-
3
4
5
6
Children have very high rates of seropositivity for both EBV and CMV.“’ Although sero ositivity for HHV6 also is common in this age group, R a weak signal for HHV-6 DNA was detected in one block of RE brain specimen using PCR with liquid hybridization. Viruses have not been cultured from specimens of the patients’ blood, nor has PCR amplification of herpesvirus genes from their blood been attempted. It is, however, of interest that one patient with RE (patient no. 7, Table 2) had a history of infectious mononucleosis at the age of 3 years, 3 years before seizure onset. It is noteworthy that the signal detectable with viral probe (after PCR) was weak in comparison to control 4ssues known to contain abundant viral genes (AIDS
7
8
9
10
11
brain tissues with CMV encephalitis or primary CNS lymphoma). Thus, the PCR data are difficult to reconcile with the abundance of CMV detected in RE brain using in situ hybridization by one group.‘” Although RE is a relatively stereotyped condition by both clinical and neuropathologic criteria,‘.” we cannot rule out the possibility that the affected patients studied in different centers may have been exposed to different herpesviruses or responded to such infection in various ways. A strong signal for EBV was detected in brain tissue from a patient without lesions of encephalitis. This suggests that EBV in the CNS is not always accompanied by inflammation. The presence of a relatively small amount of viral genome within brain does not, however, rule 877
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HUMAN PATHOLOGY
TABLE 2. Summary of Results of Polymerase Chain Reaction Using Cytomegalovirus and Epstein-Barr Virus Primers and Radiolabeled Probes Types of Tissues Studied Age (1310)at Surgery
Viral Genes Present/ Absent*
FF
Paraffin
+
+ + + + +
Tr
+
TI TI
2
6
ti/7
_ + + + + +
+
5
1
Tr Ahs + Ahs Ahs Ahs 2/6
CMV
EBV
Ahs +
Ahs + TI + + Ahs Tr 5/7
Encephalitis patients
I 2 3 4 5 6 7 Mean Nonencephalitis patients 2 3
4 5 6 hlean
41 172 83 65 85 1% 114 101.4
3.5 138 2 IS 18 17 25 69
_ _ _ +
Abbreviations: FF, fresh-frozen (at -80°C); (barely visible); t, easily visible; ++, strong. * Signal assessed on autoradiogram.
_ _ _
+ Tr
TI ++ Ahs Ahs Ahs Ahs
1993)
a child with encephalitis. This virus has been implicated in several diseases of childhood, including meningitis. 41-49Based on these studies, it does not appear to be a major factor in the pathogenesis of RE. Polymerase chain reaction has been used to show the existence of viral DNA in cases of “burnt-out” herpes encephalitis that lacked immunohistochemical evidence for HSV infection.“” Applied prospectively to surgically resected brain tissue or retrospectively to archival tissue banks, this technology may provide important clues to the pathogenesis of many inflammatory disorders of the CNS. Furthermore, examination (using PCR) of malformed brain tissue not associated with a seizure disorder, tissue from patients with intractable epilepsy but no cortical malformation or encephalitis, and normal brain tissue from individuals representing a range of ages may help to clarify the association of herpesviruses with these structural lesions and with epilepsy itself. These studies are currently in progress. Acknowledgmrnl.
Diana Lenard Secor, Stephen Kaufman,
and Carol Appleton assisted with the preparation of tissues and illustrations. Dr Bruce Quinn and Sue Yudovin provided invaluable assistance with retrieval of clinical information on the patients.
2/6
Ahs, absent; Tr, trace
REFERENCES
out a role for that virus in causing
inflammatory or immune complex-mediated injury or tissue damage by other mechanisms.‘7,“2 Indirect mechanisms of CNS damage hypothesized to occur after experimental CMV infection include the secretion of inflammatory mediators from infiltrating monocytes/macrophages, vascular and astrogliosis; some of these changes compromise, may occur after most of the virus has been cleared from the brain.gg,‘4 We also conclude that PCR using freshfrozen tissue yields more consistently reproducible data (eg, in terms of intensity of the GA3PD signal) and that hybridization of PCR product with a specific radiolabeled probe markedly increases the sensitivity of the technique. This study does not conclusively prove or disprove a possible role for herpesviruses in the causation of chronic encephalitis associated with seizures. Herpesviruses simply may accumulate in the CNS with the passage of time, and the smaller number of infected specimens in the non-RE group may be a reflection of the relatively younger age of these patients at the time of surgery. Other investigators previously have suggested a scheme by which one or more of several viral infections of the CNS may lead to chronic encephalitis32 and the syndrome recognized as RE, eg, by deposition of immune complexes in the cerebral blood vessel wall. Epstein-Barr virus, in particular, has been implicated in heterogeneous forms of encephalitis in immunosuppressed children and adults as well as in primary CNS lymphoma of patients with AIDS.3”-“” Both CMV and EBV have been found (using PCR) in various tissues from patients who have undergone organ transplantation.40 We have been able to demonstrate the presence of low levels of HHV-6 genes in one brain specimen from 878
1. Rasmussen T, OlsLewski J, Lloycl-Smith D: Focal seizures due to chronic localized encephalitis. Neurology 8:435-445, 1958 2. Abwilar MJ, Rasmussen 1‘: Role of encephalitis in pathogenesis of epilepsy. Arch Neural 2:663-676, 1960 3. Rasmussen T: Further observations on the syndrome of chronic encephalitis and epilepsy. Appl Neurophysiol 4 1:l-l 2, 1978 4. (Gordon N: Chronic progressive epilepsia partialis continua of childhood: Rasmussen synclrome. Dev Med Child Neural 34: 182-I 85, 1992 5. Gray F, Serdar-u M, Baron H, et al: Chronic localised encephalitis (Rasmussen’s) in an adult with epilepsia partialis continua. J Neural Neurosurg Psychiatry 50:747-75 1, 1987 6. Piatt JH, Jr, Hwang PA, Armstrong DC, et al: Chronic focal encephalitis (Rasmussen syndrome): Six cases. Epilepsia 29:268-279. 1988 7. Zupanc ML, Handler EC;, Irvine RL, et al: Rasmussen ew cephalitis: Epilepsia partialis continua secondary to chronic encephalitis. Pediatr Neurol 6:397-40 1, 1990 8. C;upta PC, Roy S, Tandon PN: Progressive epilepsy due to chronic persistent encephalitis. Report of four cases. .J Neural SCi 22: 105-120, 1974 9. Cupla PC, Rapin I, Houroupian DS, et al: Smolder-ing encephalitis in children. Neuropediatrics 15: 19 l-l 97, 1984 10. Honavar M, Janata 1, Polkey CE: Rasmussen’s encephalitis in surgery for epilepsy. Dev Med Child Neural 34:3-14, 1992 1 1. Rohitaille Y: Neuropathologic aspects of chronic encephalitis, in Andermanrl F (ed): Chronic Encephalitis and Epilepsy. Rasmussen’s Syndrome. Boston, MA, Butterworth-Heinernatln, 1991, pp 79-l 10 12. Farrell MA, DeRosa MJ, Curran JC, et al: Neuropathologic findings in cortical resections (including hemispherectomies) pel-formed for the treatment of intractable childhood epilepsy. Arra Neuropathol 83:246-259, 1992 13. l’ower C, Poland SD, Blume WT, et al: Cytomegalovirus and Rasmussen’s encephalitis. Lancer 336: 1282- 1284, 1990 14. Walter (;F, Renella RR: Epstein-Barr virus in brain and Rasmussen’s encephalhis. Lancet 1:279-280, 1989 15. Walter (;F, Renella RR, Hori A, et al: Nachweis van EpsteinBarr-Viren bei Rasmussen’s Enzephalitis. Nervenarzt 60: 168-l 70, 1989 16. I.ahl R, Boenigk H, Holthausen H, et al: Hemispherectomy of three epileptic children suflering from Rasmussen’s encephalitis: Neuroparhologic and virologic results. Clin Neuropathol 1 1:285-286. 1992
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IN RASMUSSEN
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17. Farrell MA, Cheng L, Cornford ME, et al: Cytomegalovirus and Rasmussen’s encephalitis. Lancet 337:1551-1552, 1991 18. Friedman H, Ch’ien L, Parham D: Virus in brain of child with hemiplegia, hemiconvulsions, and epilepsy. Lancet 2:666, 1977
(Vinters et al)
pesvirus 6 infections in children: A prospective serologic study. J Infect Dis 165:1163-1164, 1992 32. Andrew5 JM, Thompson JA, Pysher TJ, t‘t al: Chronic encephalitis, epilepsy and cerebrovascular immune complex deposits. Ann Neural 28:88-90, 1990 33. Booss J, Dann PR, Winkler- SR, et al: Mechanisms of injury to the centl-al nervous system following experimental cytomegalovirus infection. Am J Otolaryngol 11:313-317, 1990 34. Booss J, Winkler SK, Griffith BP, et al: \‘iremia and glial nodule encephalitis after experimental systenlic cytomegalovirus infection. Lab Invest 61:644-649, 1989 35. Pedneault L, Katz BZ, Miller G: Detection of Epstein-Barr virus in brain by the polymerase chain reaction. Ann Neural 32: 1 X4192, 1992 36. Morgello S: Epsteirr-Barr and human immunodeficiency viruses in acquired immunodeficiency syndrome-related primary central nervous system Iymphoma. Am J Pathol 14 1:44 l-450, 1992 37. Ross CW, Schlegelmilch JA, Grogan TM, et al: Detection of Epstein-Barr virus genome in Ki-1 (CD30)-positive. large-cell anaplastic lymphomas using the polymerase chain react ion Am J Path01 14 1: 457-465, 1992 38. Samosruk M, Ravel J: Frequent detection of Epstein-Bar] viral deoxyribonucleic acid and absence of cytomegalovirus deoxyribonucleic acid in Hodgkin’s disease and acquired immunodeficiencF syndrome-related Hodgkin’s disease. Lab Invest 65:631-636, 1991 39. Weiss LM, Chen Y-Y, Liu X-F, et al: Epstein-Barr virus and Hodgkin’s disease. A correlative in situ hybridization and polymerase chain reaction study. Am J Pathol 139: 1259-l 265, I991 40. Rogers BB, Alpert LC, Hine EAS, el al: Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. Am ,J Pathol 136:541-548, 1990 4 1, Leach CT, Sumaya CV, Brown NA: Human herpesvirus-6: Clinical implications of a recently discovered, ubiquitous agent. J Pediatr 121:173-181, 1992 42. Pruksananonda P, Hall CR, Insel RA. ct ;aI: Primary hullian herpesvirus 6 infection in young children. N Engl J Med 326: 14451450, 1992 43. Yoshikawa T, Nakashima T, Suga S, et al: Human herpesvirus6 DNA in c-erebrospinal fluid of a child with rxanthem suhitum and meningoencephalitis. Pediatrics 89:X88-890, 1992 44. Nicoll JAR, Maitland NJ, Love S: Autopsy neuropathological findings in ‘burnt out’ herpes simplex encephalitis and use of the Appl polymerase chain reaction to deter t viral DUA. Ueuropathol Neurobiol 17:3i5-382, 1991
19. Vinters HV, Fisher RS, Cornford ME, et al: Morphological substrates of infantile spasms: Studies based on surgically resected cerebral tissue. Childs Nerv Syst 8:8-l 7, 1992 20. DeRosa MJ, Secor DL, Barsom M, et al: Neuropathologic findings in surgically treated hemimegalencephaly: Immunohistochemical, morphometric, and ultrastructural study. Acta Neuropathol 84:250-260, 1992 2 1. Hayward JC, Titelbaum DS, Clancy RR, et al: Lissencephalypachygyria associated with congenital cytomegalovirus infection. J Child Neural 6:109-114, 1991 22. Byrne PJ, Silver MM, Gilbert JM, et al: Cyclopia and congenital cytomegalovirus infection. Am J Med Genet 28:61-65, 1987 23. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989 24. Mullis KB: The unusual origin of the polymerase chain reac lion. Sci Am 262:56-65, 1990 25. Mullis KB, Faloona FA: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335350. 1987 26. Impraim CC, Saiki RK, Erlich HA, et al: Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun 142:710-716, 1987 27. Shibata D, Martin WJ, Arnheim N: Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: A bridge between molecular biology and classical histology. Cancer Res 48:45644566, 1988 28. Shibata D, Klatt EC: Analysis of human immunodeficiency virus and cytomegalovirus infection by polymerase chain reaction in the acquired immunodeficiency syndrome. Arch Pathol Lab Med 1 13: 1”59-1244, _. 1989 29. Greer CE, Peterson SL, Kivia NB, et al: PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. AIMJ Clin Pathol 95:l 17.124, 1991 30. Medearis DN. Jr: Cytomegalovirus, and Plotkin SA: Infectious mononucleosis, in Behrman RE, Kliegman RM (eds): Nelson Textbook of Pediatrics (ed 14). Philadelphia, PA, Saunders, 1992, pp 803-808 31. Huang I.-M, Lee C-Y, Chen J-Y, et al: Primary human her-
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atrophy, scarring, and chronic inflammation manifesting as microglial nodules and perivascular cuffing by mononuclear inflammatory cells, with microvascular proliferation. ‘-I ’The cortex and white matter may be affected to variable degrees.‘” The definitive establishment of a specific viral etiology for RE has proven elusive. However, over the past few years in situ hybridization has been used to show that cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV) may be found within the inflamed central nervous system (CNS) tissue,‘“-‘” although using comparable methodology these pathogens have not been found in all resected RE tissues.” Additionally, enterovirus-like particles have been found by electron microscopy in a child with the related syndrome of hemiplegia, hemiconvulsions, and epilepsy. Ix We extended the search for a specific viral etiology fat RE and chronic encephalitis associated with epilepsy using the sensitive technique of polymerase chain reaction (PCR) on resected brain tissue.
Herpesviruses (especially cytomegalovirus [CMV] and Epstein-Barr virus [EBV]) have been implicated in the pathogenesis of chronic (Rasmussen) encephalitis associated with epilepsy. To assess the presence of herpesvirus genes in brain tissue from epileptic children with chronic (usually Rasmussen type) encephalitis, DNA was extracted from surgically resected brain tissue and studied by the polymerase chain reaction using primers specific for CMV, varicella zoster virus, herpes simplex virus, EBV, and human herpesvirus 6 genes. By this technique evidence for the presence of low levels of CMV and EBV genes was detected in most brain specimens from encephalitis patients and in several brain specimens from patients without encephalitis (eg, cortical dysplasia, gliosis, or encephalomalacia) who also had intractable epilepsy. Occasionally, both EBV and CMV genes were found in the same brain. Signal strength for both CMV and EBV was much lower in epileptic brains than in the brains of acquired immunodeficiency syndrome patients with CMV encephalitis or brain lymphoma. We found evidence for infection of the brain by human herpesvirus 6 in only one patient with encephalitis. Polymetase chain reaction technology applied to resected brain tissue from epileptic patients may provide evidence for or against viral-mediated pathogenesis of Rasmussen encephalitis or other types of encephalitis. The small amounts of EBV and CMV genes found suggest that herpesvirus infection of the brain does not directly cause Rasmussen encephalitis. HUM PATHOL 24:871879. Copyright 0 1993 by W.B. Saunders Company
MATERIALS
AND METHODS
Study material
Among recognized neuropathologic substrates of intractable focal epilepsy in children is a form of chronic encephalitis characterized initially by Rasmussen and colleagues.‘.” Clinically, Rasmussen-type encephalitis (RE) presents with uncontrollable unilateral focal seizures or epilepsia partialis continua associated with a progressive neurologic deficit (usually hemiparesis sometimes advancing to hemiplegia), with variable intellectual impairment. ‘-’ Resection of the affected brain tissue may ameliorate the condition.“-“’ The resected brain tissue shows features of a chronic encephalitis with
who undetwetit
included brain tissues frown seven children I esections for epilepsy extensive focal cortical
Six patients presented with typical clinical Ceatures suggestive of RE. One child had a poorly documented history of encephalitis at the age of 2 years with subsequent onset of an intractable seizure disorder. The mean age of symptom onset in the seven patients was 48.6 months (range, 24 to 72 months), while the mean age at surgery was 10 I .4 months (range, 4 1 to 172 months), yielding a mean interval of disease onset to sul-gery of 52.8 months (range, 8 to 1 14 months). The typical histopathologic features of RI?‘,“,‘” were seen in six patients (Fig 1); the child with a questionable antecedent history of encephalitis at the age of 2 years had the additional unusual finding of numerous multinucleatecl giant c,ells within the resected tissue (Fig 1C). A detailed report on the light microscopic, ultrastructural, and inimurlohistochemical features (emphasizing macrophage, microglial, major histocompatihility complex, and lymphocyte markers) of the resected tissue is in preparation. Control tissues from which DNA was rsolated included (I) paraffin sections derived from pellets of cultured glial or fibroblast cells (either noninfected or infected with CMV, varicella roster virus [VZV], or HSV); (2) sections of pelleted Iymphoblastoid cell lines either noninfected or infected with human herpesvirus 6 (HHV-6); (3) paraffin sections of pellets of R+ji cells (American Type Culture Collection, Rockville, MD) derived from human Rurkitt lymphoma and known to contain ERV genome or non-EISV-infected lymphoid cells (a gift of Dr S. David Hudnall, Department of Pathology alid l>aboratory Medicine, I J<:I,A): (4) tissue con-
From the Department of Pathology and Laboratory Medicine, and the Brain Research Institute, UCLA Medical Center, Los Angeles. <:A; and the Department of Patholob7/, UC San Diego School of Medic,ine, La Jolla, CA. Accepted for publication January 1.5, 1993. Supported in part by Public Health Service (irants No. NS 26312 ,md 28383 (to H.V.V.) and by a grant from the University of California AIDS Task Force, Public Health Service &ants No. NS 25178, 27417, and 27810, and MH45294, and funds from the Robert H. Fer-nrr Fund (to C.A.W.). KI,~ wor&: chronic (Rasmussen type) encephalitis, epilepsy, herpesviruses, polymerase chain reaction. Address correspondence and I-eprint requests to Harry V. Vinters, MD, Department of Pathology and I.aboratory Medicine, CHS 1% 170. UCI,A Medical Center, Los Angeles, CA 90024-1732. Copyright 0 1993 by W.B. Saunders Company 0046-8 I77:r,s!2408-ooo8$5.00/0
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1993)
FIGURE 1. Histopathologic features of chronic encephalitis associated with pediatric epilepsy. Panels A and B show features characteristic of RE. (A) Resected brain tissue (patient no. 2, Table 2) shows extensive chronic inflammation centered on microvessels and prominent astrocytic gliosis. (Hematoxylin-eosin stain; original magnification x85.) (B) Brain tissue (patient no. 3, Table 2) shows transmural chronic inflammatory cells in two microvessels, with extension of the inflammatory infiltrate into brain parenchyma. The degree of tissue injury (with attendant gliosis) is much less pronounced than in the example illustrated in panel A. (Hematoxylineosin stain; original magnification X560.)
trols, including paraffin sections of autopsy brain from paCents with the acquired immunodeficiency syndrome (AIDS) and known CMV encephalitis or primary CNS lymphoma that 1~36positive for EBV; and (5) resected brain tissue from children (of approximately comparable age) with intractable seizures who had focal cortical dysplasia, heminiegalencrphaly, or other tiialformati~ejtlegenerative lesions that were lacking ;I major inflattltttatot.~ cotttpotlettt.‘g.“‘~~” The lattet control group seemed appropriate Biven that certain cerebral tnalfot.tnations (eg. pa~hygyrta-lissetl~~phaly) havr been associated with herpesvirus infections, such as COIIgenital CICIV infection.“.‘” Careful review of‘ brain sections from control gmup no. 5 revealed negligible or absent evidence for chronic inflatttmation in all cases. It should be noted, howevet-, that the children with nonencephalitic lesions underwent surgery at ;I somewhat younger age than those with encephalitis (6!) u 101.4 months). The neuropathologic change in brain tissue from this group has been described in detail.“~“‘~”
III all cases genomic DNA was isolated from paraffin sections of brain using slight tnodifications of established techniques.” For two patients witlt RE and five patients with nonintlattit~iatot-y cortical lesions, fresh-frozen tissue (P800C from the time of surgery) was available for study. Briefly, paraffin sections (20 X 10 ~111thickness) were placed in a I .5-ml. Ep},etldorftrti~rofi~ge tube and treated with Histoclear to dissolve the paraffin; this material was centrifuged and the pellet was treated with 100% ethanol, which was discarded following centrifugation. From this point the material initially derived from the tissue pellet and the freshly homogenized (in .50 mmol/I, Tris-HCI) brain wrre treated in at1 identical fashion. Tissue was digcstcd Fvith proteinase K (final concentration, 200 p(g/ml,) in a digestion buffer containing 50 mmol/l. Tris (pH 8.5), I mmol/l. cthylcnediatnine tetraaretic acid, 0.5% to 1 .O% sodiuttl dodec\;l sulfate, and 0.1 or 0.05 N NaCI at 55°C for 3 horn-s. Proteinase K was inactivated by heating the tubes for 30 minutes at 05°C:. The supernatatlt was treated with 5 mol/l 1K ac~tatc and subsequently with 7.5 mol/L NH., acetate
872
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(Vinters et al)
and 100% ethanol for at least 2 hours at --8O’C. ‘The DNA pellet was resuspended in Tris-ethylenediamine tetraacetic acid, pH 7.8, and OD,,, was measured in a spertrophotometer. Although DNA yields were quite variable from case to case, 250 ~1~ of a 10% wt/vol fresh brain tissue homogenate 01 20 X 10 Fin-thick paraffin sections usually yielded a final DNA concentration of 0.25 to 1 .O pg/pL. Approximately 0.5 to 1 .O pg of DNA was used as the starting material for each PCR reaction. To minimize cross-contamination DNA isolations were carried out in a room separate from that used for preparation of the final PCR incubation mixtures and all PCR specimen mixtures were prepared in a laminar tlow hood. Isolated DNA was assayed for the presence of genes specific to five different human herpesviruses using primer pairs as shown in Table 1. Isolated DNA also was simultaneously or sequentially amplified for a gene encoding glyceraldehyde 3phosphate dehydrogenase (GASPD) as a “housekeeping” gene to assess the integrity of DNA from each specimen. Polymerase chain reaction was carried out in a Perkin Elmer Cetus (Norwalk, CT) DNA thermal cycler. Each 50-PL rnixture subjected to PCR included 0.5 to 1 .O pg of template DNA from human brain or cultured cell blocks, 30 pmol of each primer pair, 2.5 mmol/L MgC&, and 200 ~~nol/L, dNTP. Thermal cycling was carried out as follows: one cycle of 94°C (5 minutes) and 65°C (2 minutes), 30 cycles of 94°C (2 minutes) and 65°C (2 minutes), and one cycle of 72°C (7 minutes). Polymrrase chain reaction products were electrophoresed on an 8% polyacrylamide gel, which was then stained with ethidium bromide and photographed with W transillumination. For CMV. EBV, and HHV-6, specific viral gene probes complementary to the amplified DNA product were end-labeled with “P adenosine triphosphate using T4 polymerase kinase and hybridized to the PCR product after denaturation of the DNA by boiling.“” Activity of the specific probe was usually between 1.5 X 10’ and 1.5 X 10” cpm/sample. The hybridization mixture was electrophoresed through an 8% polyacrylamide gel, autoradiograms of which were then obtained at brief (2- to ?-hour) or overnight exposures at -80°C.
RESULTS FIGURE 1. (Continued) (C) Tissue from patient no. 5 (Table 2) shows marked astrogliosis and perivascular inflammation and the unusual feature of occasional perivascular multinucleated cells (arrow). The latter feature was only noted in one patient and is distinctly unusual in RE. (Hematoxylin-eosin stain; original magnification X220.)
TABLE (kwe to he Atnplitied
1.
In all cases a single visible band of variable intensity was identified on ethidium bromide-stained gels of PCR products obtained with GA3PD-specific primers, indicating the presence of adequate DKA. The band always
Herpesvirus Primer Pairs Used in Polymerase
Chain Reaction Silt3 of t:wpwted
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‘f(;GTAT[:~I‘(;(;AAC;(;A(:7C:A1‘(;A(: A’T(;CCA(;TCA(;(:TT(:(:(:(;TTCA(;<: C(:PIAGC(:(;(:CTC:TGAT,~(:(:rZ4(;C(: (:AC;(:AC<:A.I‘CCTCC:T<:TT~:~I‘<:T(;C; (:(;T(:ACAI‘ATTAT(;C:AAA(:AT(; (:(;7Tm~rA~ACA~~[~C[:(;<: C.~~ICA(:(:CAC:C:(:G(:A(;AG(;C;r\(: (;(;<;CCA(;G(:C;~~t~‘l’~~(;(;T(;~I-~~ T(~I‘(~I‘GA(:(;4GGC;C;CCA(;(:TA(:A(;<;~: (;(:A(:(:CPUZTCCAA(:TTG(;A(:C;~TTT(; (;‘I’C;AT<;TAC:CT<;(;(:(:(;T~I‘(:C’I‘(; <;.4T(:CAT<;(;T(:(;T[~~fT~;~:,4(:(;
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Volume 24, No. 8 (August
HUMAN PATHOLOGY
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1993)
89
-
271 194 118
-
603
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271 194
-
118
-
FIGURE 2. Ethidium bromide-stained polyacrylamide gels of PCR products obtained after amplification of DNA from human brain tissue. The top panel represents results of PCR using primer pairs for GA3PD gene and the lower panel represents results of PCR with primer pairs specific for CMVMIE genes. M, molecular weight standards (no. of base pairs); -, negative control, DNA from noninfected U373 cells; f, positive control, DNA from AD169 CMV-infected U373 cells. Samples included brain specimens from patients with encephalitis (E lanes), nonencephalitis (epilepsy) cortical resections (D lanes), and AIDS (autopsy specimen) with CMV encephalitis (lane A). Note the variation in intensity of the amplified GA3PD products despite loading approximately equivalent amounts of DNA in each PCR mixture. The only detectable amplified CMV product is in the control infected cells and brain tissue from the AIDSKMV encephalitis patient. All starting material for DNA extractions in this study was paraffin embedded.
was more intense using DNA derived from freshly harvested (and frozen) rather than paraffin-embedded brain tissue. DNA extracted from paraffin sections of cultured cells infected with CMV, VZV, HHV-6, or HSV, or from sections of Raji cells yielded intense bands on ethidium bromide-stained gels of PCR products at the expected molecular weight (Figs 2 to 4). Polymerase chain reaction products using template DNA obtained from epilepsyderived brain tissue and assessed by ethidium bromide failed to show the presence of CMV, HSV, VZV, HHV6, or EBV genes in the brain tissue (Figs 2 to 4). In contrast, using this method CMV genes were easily detected in brains from AIDS patients with CMV encephalitis (Fig 2). Polymerase chain reaction carried out on AIDS-related CNS lymphoma tissue using EBV primers yielded distinct bands at the expected molecular weights in over 50% of the cases (unpublished data); representative cases only are shown in Fig 4. The use of ‘2P-radiolabeled specific probes and autoradiography to detect the labeled gene significantly increased the sensitivity of detecting the PCR product. Using this technique trace or weak signals for CMV were found in at least one sample from six 874
of seven cases of encephalitis (Fig 5). The signal, however, was faint in most of these specimens even after prolonged exposure of the autoradiogram and use of an intensely labeled specific probe. In contrast, identically prepared autopsy tissue from AIDS patients with known CMV encephalitis, even of a mild degree with rare identifiable inclusions, yielded an intense band on autoradiography, essentially comparable to that seen with cell culture controls. It was of interest that brain specimens from two of six noninflammatory lesions also showed CMV, yielding a signal comparable in intensity to that noted in tissue from patients with encephalitis. Similar observations were made with EBV primers and probes. Trace or weak EBV-specific bands were noted on autoradiograms using brain tissue derived from five of seven patients with encephalitis and in two of six nonencephalitis lesions. It is noteworthy, however, that the most intense EBV-specific band was repeatedly found in the resected brain of a patient who did not show lesions of RE, but had severe hydrocephalus with secondary gliotic and destructive lesions in the cerebral cortex (Fig 6). Results from all specimens are summa-
HERPESVIRUSES
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603
(Vinters et al)
-
+
A
1
2
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-
2710 194118FIGURE 3. Ethidium bromide-stained gels of PCR products (DNA extracted from paraffin sections) using primers for GA3PD (top panel) and VZV (bottom panel). The lanes represent tissue from various sources (see Fig 2). The VZV gene is detectable only in the cultured infected cells.
603271194118-
rized in Table 2. Note that several patients showed evidence of both CMV and EBV in the brain by PCR. A weak signal suggestive of the presence of HHV6 was found after liquid hybridization of DNA extracted from only one of several paraffin tissue blocks from a single patient with RE; the radioactive signal was noted only after prolonged exposure of the autoradiogram (data not illustrated).
RE.“‘.“’ In view of our previous inabilitv to detect CMV in RE brain specimens by in situ hybridization,” we felt it important to extend our observations using highly sensitive PCR methodology. Even though we have not used quantitative PCR our results suggest that low levels of EBV and CMV genes may be found in resected brain specimens that show neuropathologic features of encephalitis, as well as in some specimens from epileptic patients without encephalitis (eg, with changes of cortical dysplasia OI other types of secondary destructive lesion). Considering our negative in situ hybridization studies with CMV PCR seems to be more sensitive for detecting probe,” small amounts of viral DNA, although the purpose of the present investigation was not a direc.t comparison of the sensitivity of in situ hybridization versus PCR. Given the sensitivity of PCR for detecting small numbers of gene copies, we cannot rule out the possibility that we have amplified blood-borne viral genes in the PCR reaction; indeed, there is no way to rule this out in the present experimental paradigm.
DISCUSSION The highly sensitive technique of PCR’4,‘” can be used to detect minute quantities of numerous genes in human tissues, whether derived from autopsy or biopsy and regardless of whether the template DNA is derived from paraffin blocks or from freshly isolated tissues.“‘-*” In this initial study of viral genes within the resected brain of patients with intractable seizures secondary to chronic (usually Rasmussen type) encephalitis, we have chosen to emphasize herpesviruses because of several reports suggesting that CMV and EBV may be of pathogenetic significance in the causation of 875
M
603
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FIGURE 4. Ethidium bromide-stained gels of PCR products obtained using DNA from the surgically resected brain tissues of a 3-month-old child with severe cortical dysplasia causing epilepsy (lane D), of four patients with histologically verified encephalitis (E lanes), and of four representative patients (autopsy tissue) with CNS lymphoma (L lanes). The top panel represents the results of PCR performed using primer pairs for a GABPD gene and the bottom panel represents the results of PCR using primer pairs for the IR3 region of the EBV genome. All starting material for this study was paraffin-embedded tissue. M, molecular weight standards.
1180
603271
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194118 -
m
-+
1
872603-
271-
FIGURE 5. An autoradiogram (2-hour exposure) of gel representing electrophoresed PCR products (using CMV primer pairs) hybridized with a 32P-labeled CMV probe. The lanes are as follows: m, molecular weight standards; -, negative cell culture control; and +, positive ceil culture control. Lanes I, 2, 3, and 8 to 12 represent PCR using DNA from histologically proven encephalitis cases; lanes 4, 5, and 7 represent PCR using DNA from nonencephalitis epilepsy cortical resections; and lane 6 represents PCR using an autopsy brain specimen from an AIDS patient with CMV encephalitis (positive tissue control). Lanes 1 and 2 represent DNA extracted from two different paraffin blocks from the same patient. All lanes except lanes 3 to 5 represent DNA isolated from paraffin sections; the DNA in lanes 3 to 5 was from fresh-frozen tissue. A faint signal was detected in lanes 9 and 10. After overnight exposure, signal was noted in lanes 1 and 7 to IO.
HERPESVIRUSES
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IN RASMUSSEN
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123456789
10
11
12
872603FIGURE 6. (Top) Autoradiogram (2-hour exposure) of gel representing PCR products (using EBV probe) hybridized with a radiolabeled probe. The apparent molecular weight of hybridized product is approximately 600 base pairs. The lanes are the same as in Fig 5, except that lane 6 contains PCR product from an AIDS patient with CNS lymphoma (positive tissue control) and lanes 7 and 8 are reversed (ie, lane 7 is from a patient with encephalitis and lane 8 is from a patient with cortical dysplasia). Note the detectable bands in lanes 4 and 7 to 11. After overnight exposure (a portion of the autoradiogram is shown in the bottom panel) signal was noted in lanes I, 4, and 7 to 11, in addition to the control lanes. The most prominent signal is in lane 4, representing resected brain tissue from an epileptic patient without features of encephalitis
271-
194-
3
4
5
6
Children have very high rates of seropositivity for both EBV and CMV.“’ Although sero ositivity for HHV6 also is common in this age group, R a weak signal for HHV-6 DNA was detected in one block of RE brain specimen using PCR with liquid hybridization. Viruses have not been cultured from specimens of the patients’ blood, nor has PCR amplification of herpesvirus genes from their blood been attempted. It is, however, of interest that one patient with RE (patient no. 7, Table 2) had a history of infectious mononucleosis at the age of 3 years, 3 years before seizure onset. It is noteworthy that the signal detectable with viral probe (after PCR) was weak in comparison to control 4ssues known to contain abundant viral genes (AIDS
7
8
9
10
11
brain tissues with CMV encephalitis or primary CNS lymphoma). Thus, the PCR data are difficult to reconcile with the abundance of CMV detected in RE brain using in situ hybridization by one group.‘” Although RE is a relatively stereotyped condition by both clinical and neuropathologic criteria,‘.” we cannot rule out the possibility that the affected patients studied in different centers may have been exposed to different herpesviruses or responded to such infection in various ways. A strong signal for EBV was detected in brain tissue from a patient without lesions of encephalitis. This suggests that EBV in the CNS is not always accompanied by inflammation. The presence of a relatively small amount of viral genome within brain does not, however, rule 877
Volume 24, No. 8 (August
HUMAN PATHOLOGY
TABLE 2. Summary of Results of Polymerase Chain Reaction Using Cytomegalovirus and Epstein-Barr Virus Primers and Radiolabeled Probes Types of Tissues Studied Age (1310)at Surgery
Viral Genes Present/ Absent*
FF
Paraffin
+
+ + + + +
Tr
+
TI TI
2
6
ti/7
_ + + + + +
+
5
1
Tr Ahs + Ahs Ahs Ahs 2/6
CMV
EBV
Ahs +
Ahs + TI + + Ahs Tr 5/7
Encephalitis patients
I 2 3 4 5 6 7 Mean Nonencephalitis patients 2 3
4 5 6 hlean
41 172 83 65 85 1% 114 101.4
3.5 138 2 IS 18 17 25 69
_ _ _ +
Abbreviations: FF, fresh-frozen (at -80°C); (barely visible); t, easily visible; ++, strong. * Signal assessed on autoradiogram.
_ _ _
+ Tr
TI ++ Ahs Ahs Ahs Ahs
1993)
a child with encephalitis. This virus has been implicated in several diseases of childhood, including meningitis. 41-49Based on these studies, it does not appear to be a major factor in the pathogenesis of RE. Polymerase chain reaction has been used to show the existence of viral DNA in cases of “burnt-out” herpes encephalitis that lacked immunohistochemical evidence for HSV infection.“” Applied prospectively to surgically resected brain tissue or retrospectively to archival tissue banks, this technology may provide important clues to the pathogenesis of many inflammatory disorders of the CNS. Furthermore, examination (using PCR) of malformed brain tissue not associated with a seizure disorder, tissue from patients with intractable epilepsy but no cortical malformation or encephalitis, and normal brain tissue from individuals representing a range of ages may help to clarify the association of herpesviruses with these structural lesions and with epilepsy itself. These studies are currently in progress. Acknowledgmrnl.
Diana Lenard Secor, Stephen Kaufman,
and Carol Appleton assisted with the preparation of tissues and illustrations. Dr Bruce Quinn and Sue Yudovin provided invaluable assistance with retrieval of clinical information on the patients.
2/6
Ahs, absent; Tr, trace
REFERENCES
out a role for that virus in causing
inflammatory or immune complex-mediated injury or tissue damage by other mechanisms.‘7,“2 Indirect mechanisms of CNS damage hypothesized to occur after experimental CMV infection include the secretion of inflammatory mediators from infiltrating monocytes/macrophages, vascular and astrogliosis; some of these changes compromise, may occur after most of the virus has been cleared from the brain.gg,‘4 We also conclude that PCR using freshfrozen tissue yields more consistently reproducible data (eg, in terms of intensity of the GA3PD signal) and that hybridization of PCR product with a specific radiolabeled probe markedly increases the sensitivity of the technique. This study does not conclusively prove or disprove a possible role for herpesviruses in the causation of chronic encephalitis associated with seizures. Herpesviruses simply may accumulate in the CNS with the passage of time, and the smaller number of infected specimens in the non-RE group may be a reflection of the relatively younger age of these patients at the time of surgery. Other investigators previously have suggested a scheme by which one or more of several viral infections of the CNS may lead to chronic encephalitis32 and the syndrome recognized as RE, eg, by deposition of immune complexes in the cerebral blood vessel wall. Epstein-Barr virus, in particular, has been implicated in heterogeneous forms of encephalitis in immunosuppressed children and adults as well as in primary CNS lymphoma of patients with AIDS.3”-“” Both CMV and EBV have been found (using PCR) in various tissues from patients who have undergone organ transplantation.40 We have been able to demonstrate the presence of low levels of HHV-6 genes in one brain specimen from 878
1. Rasmussen T, OlsLewski J, Lloycl-Smith D: Focal seizures due to chronic localized encephalitis. Neurology 8:435-445, 1958 2. Abwilar MJ, Rasmussen 1‘: Role of encephalitis in pathogenesis of epilepsy. Arch Neural 2:663-676, 1960 3. Rasmussen T: Further observations on the syndrome of chronic encephalitis and epilepsy. Appl Neurophysiol 4 1:l-l 2, 1978 4. (Gordon N: Chronic progressive epilepsia partialis continua of childhood: Rasmussen synclrome. Dev Med Child Neural 34: 182-I 85, 1992 5. Gray F, Serdar-u M, Baron H, et al: Chronic localised encephalitis (Rasmussen’s) in an adult with epilepsia partialis continua. J Neural Neurosurg Psychiatry 50:747-75 1, 1987 6. Piatt JH, Jr, Hwang PA, Armstrong DC, et al: Chronic focal encephalitis (Rasmussen syndrome): Six cases. Epilepsia 29:268-279. 1988 7. Zupanc ML, Handler EC;, Irvine RL, et al: Rasmussen ew cephalitis: Epilepsia partialis continua secondary to chronic encephalitis. Pediatr Neurol 6:397-40 1, 1990 8. C;upta PC, Roy S, Tandon PN: Progressive epilepsy due to chronic persistent encephalitis. Report of four cases. .J Neural SCi 22: 105-120, 1974 9. Cupla PC, Rapin I, Houroupian DS, et al: Smolder-ing encephalitis in children. Neuropediatrics 15: 19 l-l 97, 1984 10. Honavar M, Janata 1, Polkey CE: Rasmussen’s encephalitis in surgery for epilepsy. Dev Med Child Neural 34:3-14, 1992 1 1. Rohitaille Y: Neuropathologic aspects of chronic encephalitis, in Andermanrl F (ed): Chronic Encephalitis and Epilepsy. Rasmussen’s Syndrome. Boston, MA, Butterworth-Heinernatln, 1991, pp 79-l 10 12. Farrell MA, DeRosa MJ, Curran JC, et al: Neuropathologic findings in cortical resections (including hemispherectomies) pel-formed for the treatment of intractable childhood epilepsy. Arra Neuropathol 83:246-259, 1992 13. l’ower C, Poland SD, Blume WT, et al: Cytomegalovirus and Rasmussen’s encephalitis. Lancer 336: 1282- 1284, 1990 14. Walter (;F, Renella RR: Epstein-Barr virus in brain and Rasmussen’s encephalhis. Lancet 1:279-280, 1989 15. Walter (;F, Renella RR, Hori A, et al: Nachweis van EpsteinBarr-Viren bei Rasmussen’s Enzephalitis. Nervenarzt 60: 168-l 70, 1989 16. I.ahl R, Boenigk H, Holthausen H, et al: Hemispherectomy of three epileptic children suflering from Rasmussen’s encephalitis: Neuroparhologic and virologic results. Clin Neuropathol 1 1:285-286. 1992
HERPESVIRUSES
IN RASMUSSEN
ENCEPHALITIS
17. Farrell MA, Cheng L, Cornford ME, et al: Cytomegalovirus and Rasmussen’s encephalitis. Lancet 337:1551-1552, 1991 18. Friedman H, Ch’ien L, Parham D: Virus in brain of child with hemiplegia, hemiconvulsions, and epilepsy. Lancet 2:666, 1977
(Vinters et al)
pesvirus 6 infections in children: A prospective serologic study. J Infect Dis 165:1163-1164, 1992 32. Andrew5 JM, Thompson JA, Pysher TJ, t‘t al: Chronic encephalitis, epilepsy and cerebrovascular immune complex deposits. Ann Neural 28:88-90, 1990 33. Booss J, Dann PR, Winkler- SR, et al: Mechanisms of injury to the centl-al nervous system following experimental cytomegalovirus infection. Am J Otolaryngol 11:313-317, 1990 34. Booss J, Winkler SK, Griffith BP, et al: \‘iremia and glial nodule encephalitis after experimental systenlic cytomegalovirus infection. Lab Invest 61:644-649, 1989 35. Pedneault L, Katz BZ, Miller G: Detection of Epstein-Barr virus in brain by the polymerase chain reaction. Ann Neural 32: 1 X4192, 1992 36. Morgello S: Epsteirr-Barr and human immunodeficiency viruses in acquired immunodeficiency syndrome-related primary central nervous system Iymphoma. Am J Pathol 14 1:44 l-450, 1992 37. Ross CW, Schlegelmilch JA, Grogan TM, et al: Detection of Epstein-Barr virus genome in Ki-1 (CD30)-positive. large-cell anaplastic lymphomas using the polymerase chain react ion Am J Path01 14 1: 457-465, 1992 38. Samosruk M, Ravel J: Frequent detection of Epstein-Bar] viral deoxyribonucleic acid and absence of cytomegalovirus deoxyribonucleic acid in Hodgkin’s disease and acquired immunodeficiencF syndrome-related Hodgkin’s disease. Lab Invest 65:631-636, 1991 39. Weiss LM, Chen Y-Y, Liu X-F, et al: Epstein-Barr virus and Hodgkin’s disease. A correlative in situ hybridization and polymerase chain reaction study. Am J Pathol 139: 1259-l 265, I991 40. Rogers BB, Alpert LC, Hine EAS, el al: Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. Am ,J Pathol 136:541-548, 1990 4 1, Leach CT, Sumaya CV, Brown NA: Human herpesvirus-6: Clinical implications of a recently discovered, ubiquitous agent. J Pediatr 121:173-181, 1992 42. Pruksananonda P, Hall CR, Insel RA. ct ;aI: Primary hullian herpesvirus 6 infection in young children. N Engl J Med 326: 14451450, 1992 43. Yoshikawa T, Nakashima T, Suga S, et al: Human herpesvirus6 DNA in c-erebrospinal fluid of a child with rxanthem suhitum and meningoencephalitis. Pediatrics 89:X88-890, 1992 44. Nicoll JAR, Maitland NJ, Love S: Autopsy neuropathological findings in ‘burnt out’ herpes simplex encephalitis and use of the Appl polymerase chain reaction to deter t viral DUA. Ueuropathol Neurobiol 17:3i5-382, 1991
19. Vinters HV, Fisher RS, Cornford ME, et al: Morphological substrates of infantile spasms: Studies based on surgically resected cerebral tissue. Childs Nerv Syst 8:8-l 7, 1992 20. DeRosa MJ, Secor DL, Barsom M, et al: Neuropathologic findings in surgically treated hemimegalencephaly: Immunohistochemical, morphometric, and ultrastructural study. Acta Neuropathol 84:250-260, 1992 2 1. Hayward JC, Titelbaum DS, Clancy RR, et al: Lissencephalypachygyria associated with congenital cytomegalovirus infection. J Child Neural 6:109-114, 1991 22. Byrne PJ, Silver MM, Gilbert JM, et al: Cyclopia and congenital cytomegalovirus infection. Am J Med Genet 28:61-65, 1987 23. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989 24. Mullis KB: The unusual origin of the polymerase chain reac lion. Sci Am 262:56-65, 1990 25. Mullis KB, Faloona FA: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335350. 1987 26. Impraim CC, Saiki RK, Erlich HA, et al: Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun 142:710-716, 1987 27. Shibata D, Martin WJ, Arnheim N: Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: A bridge between molecular biology and classical histology. Cancer Res 48:45644566, 1988 28. Shibata D, Klatt EC: Analysis of human immunodeficiency virus and cytomegalovirus infection by polymerase chain reaction in the acquired immunodeficiency syndrome. Arch Pathol Lab Med 1 13: 1”59-1244, _. 1989 29. Greer CE, Peterson SL, Kivia NB, et al: PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. AIMJ Clin Pathol 95:l 17.124, 1991 30. Medearis DN. Jr: Cytomegalovirus, and Plotkin SA: Infectious mononucleosis, in Behrman RE, Kliegman RM (eds): Nelson Textbook of Pediatrics (ed 14). Philadelphia, PA, Saunders, 1992, pp 803-808 31. Huang I.-M, Lee C-Y, Chen J-Y, et al: Primary human her-
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