Induced Meningoencephalomyelitis in Baboons

Outbreak of Orthoreovirus-Induced Meningoencephalomyelitis in Baboons

M. Michelle Leland,1 Gene B. Hubbard,2 Henry T. Sentmore III,3 Kenneth F. Soike,4 and Julia K. Hilliard3

Background and Purpose: Spontaneous viral encephalitis is rare in the baboon; yet, during a 13-month period (1993–1994), eight juvenile baboons (Papio cynocephalus spp.) developed acute, progressive nonsuppurative meningoencephalomyelitis caused by an unknown agent. Clinical signs of disease included disorientation and truncal ataxia that rapidly progressed to hemiparesis or paraparesis. Clinicopathologic findings were not remarkable and appreciable gross lesions were not seen at necropsy. Microscopic examination revealed CNS lesions that were characterized by lymphoplasmacytic perivascular cuffing, microglial nodules, demyelination, axonal degeneration, vacuolization, and hemorrhage. Subsequently, a novel syncytium-inducing mammalian orthoreovirus was isolated from the brain tissue of five baboons with clinical signs of infection. Methods: To confirm the etiologic role of the orthoreovirus, two juvenile baboons were inoculated with the virus, then were monitored for 6 weeks. Results: Lesions similar to those seen in spontaneous cases were found in the CNS, and orthoreovirus was isolated from the brain of both animals. Conclusion: Analysis of the outbreak indicated juvenile baboons were most susceptible to disease and the virus had a possible incubation time of 46 to 66 days, but did not indicate a source of the virus or mode of transmission.

Spontaneous viral encephalitis is seldom diagnosed in ba- nerves to sensory and motor ganglia (10-12). Variants with as boons (1, 2). Yet from January, 1993 to January, 1994, eight juve- little as a single amino-acid substitution can have altered pat- nile baboons (Papio cynocephalus spp.) housed at the Southwest terns of CNS tropism (11). Foundation for Biomedical Research (SFBR) developed clinical Reoviruses are characterized by a double-stranded RNA of 10 signs of disease that were consistent with acute, progressive linear segments with individual molecular mass between 0.5 to encephalitis. The eight animals were juveniles housed in three 3.0 x 106 Da. The G:C ratio is approximately 43:100; buoyant den- 20 by 30-ft or one 20 by 50-ft concrete-floored, one-inch galva- sity in CsCl is 1.36 g/cm2. Virions are isometric, nonenveloped, and nized chain-link gang cages that were widely separated from 60 to 80 nm in diameter. Reoviruses are resistant to treatment each other by other identical gang cages that did not house with ether and acid. Mammalian-type reoviruses agglutinate clinically affected baboons. These gang cages were in two build- human type-O red blood cells. Reoviruses 1 and 2 are uniform, ings of essentially identical gang cages, which housed baboons whereas reovirus 3 has multiple subtypes (13). of mixed age and sex, with most of them being juveniles. Each We report an outbreak of viral encephalitis in group-housed cage housed 25 or fewer baboons at any time (Fig. 1). juvenile baboons at the SFBR. Because this report follows the Virus isolation from brain tissues from the affected baboons isolation and identification of the viral agent, the emphasis is yielded a novel syncytium-inducing orthoreovirus (3). Viruses of on clinical signs of disease, pathologic changes, and manage- this genus are usually nonpathogenic, except in rodents and ment leading to identification of the etiologic agent, treatment, and poultry (4). Orthoreoviruses have been studied intensively with epidemiology of the first orthoreovirus to be isolated from baboons. respect to CNS infections in mice (5). Sabin observed that after experimentally induced infection by intracranial inoculation, Materials and Methods monkeys developed meningitis, but not encephalitis (6). There The eight affected baboons were members of a biomedical re- have been reports of reovirus-associated encephalitis in hu- search colony of approximately 3,000 baboons. The affected ba- mans (7), but data are limited. In rare instances, reovirus has boons were housed and became ill in three 20 by 30-ft and one been implicated as a primary etiologic agent of respiratory tract 20 by 50-ft one-inch galvanized chain-link gang cages that were disease in nonhuman primates but, to the authors’ knowledge, widely separated from each other in two buildings of essentially has not be reported in the baboon (8). There also has been a identical gang cages. Figure 1 shows the locations of the af- speculative association with hepatitis and extrabiliary atresia fected baboons and the dates of diagnosis. The animals became in the macaque (9). ill while housed in the outdoor gang cages, and all had been ex- Under natural conditions, reoviruses enter the host through posed to other baboons that became ill. Seven of the baboons the respiratory and gastrointestinal tracts then spread either were housed in the same gang cage (104.18). Two were trans- hematogenously (5) or in retrograde fashion through peripheral ferred to the hospital where one died and the other was euthanized. The other five were transferred to two separate Departments of 1Physiology and Medicine, 2Laboratory Animal Medicine, and 3Virology and Immunology, Southwest Foundation for Biomedical Research, San cages (104.12 and 104.15), then were hospitalized and euthanized Antonio, TX; and 4Department of Microbiology, Tulane Regional Primate Re- (Table 1). One baboon was never housed in cage 104.18, but search Center, Covington, LA. 199 Vol 50, No 2 Comparative Medicine April 2000

Figure 1. Dates and locations of baboon orthoreovirus (BRV) clinical cases. shared cage 104.15 with three other baboons that became ill. Louis encephalitis virus; western and eastern equine encephalitis Prior to being housed in cages 104.18 and 104.15, the baboons virus; dengue virus 2; rubeola virus; rubella virus; arbovirus pools had not been housed together. These outdoor gang cages housed (groups A and C, California encephalitis and Bunyamwera, respec- approximately 25 baboons of mixed age and either sex, with fre- tively); mumps virus; simian T-lymphotropic virus 1; simian immu- quent transfer of individual baboons throughout the SFBR (Fig- nodeficiency virus; simian retroviruses 1 and 2; foamy virus; ure 1). Fewer than 50 baboons are added to the colony from lymphocytic choriomeningitis virus; reoviruses 1–3; cercopithecine outside sources each year. virus type 1; Herpesvirus papio 2; encephalomyocarditis virus; his- Baboons were housed in individual metal baboon cages in a toplasmosis; coccidioidomycosis; blastomycosis; and cryptomycosis. climate-controlled AAALAC-approved facility after clinical The CSF samples taken from four of the affected baboons and one signs of disease developed. Animal care and experimentation control baboon who did not have clinical signs prior to death were was conducted in accordance with all relevant local, state, and cultured for bacteria. national regulations, using protocols approved by the appropri- Six of the affected baboons were given supportive treatment ate Institutional Animal Care and Use Committees. All were fed for dehydration and low doses of dexamethasone (VEDCO, St. commercial primate food (Teklad 15% Monkey Diet (W), Joseph, MO) once a day (2 to 3 mg given intramuscularly [IM]) Harlan, Madison, WI), and water was available ad libitum. Ani- and chloramphenicol sodium succinate, USP (Lymphomed, mals were euthanized by intravenous administration of sodium Deerfield, IL) twice a day (25 mg/kg of body weight, IM) to alle- pentobarbitol solution (Fort Dodge Laboratories, Fort Dodge, IA). viate signs of suspected meningitis. Two animals did not receive Clinical evaluation and treatment: Clinical records did treatment. Table 1 provides a summary of the clinical and histo- not indicate previous history of disease in any of the affected logic observations and virus isolation for each baboon. animals. When possible, body weight and temperature, and Pathologic examination: Thorough necropsies were per- complete blood count (CBC), cerebral spinal fluid (CSF) analy- formed on all eight baboons, two baboons infected experimen- sis, and serum biochemical analysis results were determined for tally, possibly infected baboons, seven feral rats from the area from affected baboons. Additionally, serum samples were ob- where the affected baboons were housed, and mice experimen- tained and stored at -70ЊC for subsequent antibody screening tally infected in the diagnostic studies. Spinal cord was not har- against selected viral and fungal agents. Sera also were ob- vested from the early cases. Tissues were preserved in tained from 20 control baboons that did not manifest clinical neutral-buffered 10% formalin, processed conventionally, em- signs of disease but were housed in the same gang cage area as bedded in paraffin, cut into 5-␮m-thick sections, and stained the affected animals. Many selected sera and tissues from ba- with hematoxylin and eosin (H&E). Special stains were used as boons with clinical signs of infection, possibly infected baboons, indicated. Brain tissue from three of the affected baboons, possi- and baboons that were considered to be healthy controls were sub- bly infected baboons, and several baboons that died of other mitted to various laboratories in addition to the SFBR and Tulane causes was cultured for possible bacterial infection. Additional Regional Primate Research Center for screening for a possible etio- tissues, serum, and CSF from encephalitic, experimental, and logic agent. These samples were tested for polioviruses 1–3; St. control baboons, feral rats, possibly infected baboons, and ex- 200 Orthoreovirus Meningoencephalomyelitis in Baboons

perimentally infected mice were harvested and kept frozen at ies were not detected in neutralization assays, but polyclonal -70ЊC. Tissues were tested for bacteria when indicated. antibodies were detected in immunofluorescence assays, using a Histologic review of all spontaneous SFBR baboon encephali- modified enzyme-linked immunoassay (ELISA) and Western tis cases accumulated over the prior 10 years was done for mor- blot techniques in conjunction with the virus isolate. The latter phologically similar cases. assays were based on use of whole-infected cell lysates from the Virus isolation: Brain tissue from five baboons with clinical BRV 10895, as well as mock-infected lysates from Vero cells in signs of CNS disease and four clinically normal baboons was culture (3). Serum samples obtained from the retrospective ba- used as the standard tissue for virus isolation. The CSF from boon cases that had morphologic lesions consistent with those of three baboons with clinical signs of disease was cultured di- the current eight cases also were evaluated for antibodies. rectly for detection of virus. Heart, lung, liver, kidney, adrenal Twenty baboons housed in the same cages that did not have gland, spleen, urinary bladder, testis, lymph node, pancreas, clinical signs of disease also were evaluated. stomach, small and large intestines, axillary nerve, spinal cord, Experimental mouse study: In an effort to document and whole blood also were obtained. These samples were inocu- neurovirulence and learn more about the reovirus, an experi- lated as supernatants of approximately 10% homogenates onto mental mouse study was done. Twenty-five Swiss Webster a variety of mammalian cells in culture maintained in a 5% C02 Hsd:ND4 suckling outbred mice (Harlan Sprague Dawley, In- environment at 37ЊC. Cell cultures of primary rhesus monkey dianapolis, IN) were allotted to five groups and were given ei- kidney, HEp-2, mouse L, MA104, QT6, and Vero cells were used ther 30 ␮l of BRV 10895-infected Vero cells (group 1), 10% for virus isolation. Inoculated plates were incubated for 10 days infected brain suspension prepared from baboon 10895 (group and were observed daily for appearance of cytopathic effect 2), uninfected Vero cells (group 3), control brain from unaffected (CPE). If negative for CPE after 10 days, cell culture superna- animals (group 4), or control medium (group 5). All suspensions tant was passaged at least once and cells were kept for an addi- were prepared in sterile normal saline. One sentinel mouse was tional 10 days. If CPE were not observed after that period, placed in the same cage with each group. Mice were euthanized samples were considered to be virus negative. Virus isolated with CO2 gas when they appeared ill and unable to sustain from the brain of baboon 10895 was selected as the representa- daily food intake. tive strain of the baboon orthoreovirus (BRV) group of isolates Experimental baboon study: Two apparently healthy fe- and was termed BRV 10895. male juvenile baboons, aged 20 and 22 months, with no previous Electron microscopy: Brain, spinal cord, and infected Vero history of signs of CNS dysfunction were sent from the SFBR to cells from baboon 10895 as well as control Vero cells were evalu- the Tulane Regional Primate Research Center for experimental ated by use of transmission electron microscopy (TEM). Speci- inoculation with BRV. The baboons were given intracranial and mens were fixed in 3.0% glutaraldehyde in 0.2M sodium intravenous doses of BRV 10895, using 105 plaque-forming cacodylate buffer (pH 7.3) at 4ЊC for 24 hours, then were washed units/ml in a volume of 0.25 ml. Intracranial doses were given overnight in buffer. Specimens for TEM were treated with os- through a small-gauge burr hole over the right temporal lobe; a mium tetroxide (1%) for 1 hour, dehydrated in graded ethanols, 1.0-ml dose was injected into the saphenous vein. These ani- and embedded in Spurr’s epoxy resin. Semi-thin (1 ␮m) and thin mals were monitored daily for successful experimental trans- (90 nm) sections were cut and evaluated. Thin sections were mission of infection, tested for virus, and observed for clinical double-stained with uranyl acetate and lead citrate. Infected signs of disease over a 6-week interval. Blood samples and Vero cell culture supernatants were processed for TEM evalua- throat swab specimens were taken on arrival and prior to inocu- tion by use of the negative staining technique. Supernatant lation. After a 2-week acclimation, baboons were experimentally samples were centrifuged at 14,000 xg in an Eppendorf tube for 10 infected under aseptic conditions. Blood samples and throat swab minutes. Supernatant was removed, and cells were extracted with specimens were collected at specified times, including postinocula- an equal volume of 1,1,2-trichloro-1,1,2-trifluoroethane (Freon tion days (PID) 3, 7, 14, and 21. Animals were observed for changes 113). Ammonium molybdate (1%) was used subsequently for nega- in their behavior and were euthanized 6 weeks after inoculation. tive staining. Specimens were examined by use of a Philips 410 LS electron microscope operating at 80 kV. Results Virus identification: Virus neutralization, using polyclonal Clinical evaluation and treatment: Affected baboons immune sera, was attempted to identify reoviruses 1–3 once re- ranged in age from 10 to 44 (mean=18) months and included six sults of TEM became available. males and two females. Individual animals became sick over a Serologic testing: Five serum samples from four encephal- period of 13 months. Clinical records did not indicate previous itic baboons were evaluated for the presence of neutralizing an- history of CNS disease in any of the affected animals. The clini- tibodies against BRV 10895. Single serum samples were cal picture for the eight cases was similar. Each baboon had obtained from three affected animals during clinical illness (in- hind limb weakness, causing signs that ranged from unilateral cluding baboon 10895). Inactivated sera (30 min at 56ЊC) were weight-bearing lameness in one animal to moderate-to-severe assessed at four five-fold dilutions, with the lowest dilution be- truncal ataxia that caused the animal to fall backward in four ing 1:10. Serum was mixed with 320 TCID50 BRV 10895. After cases. Disorientation that progressed in severity over the course 2 hours at room temperature (22ЊC), mixtures were maintained of disease was observed in three baboons. Generalized Jackso- at 4ЊC overnight. Subsequently, 0.2 ml of each mixture or spe- nian epileptic seizures were seen in three of the baboons. Two cific quantities of control virus were inoculated onto separate baboons with signs of hemiparesis had head tilt on the affected Vero cell cultures and were evaluated for 7 days (3). Hemagglu- side. One animal had vertical nystagmus. Seven of the animals tinating capacity of BRV 10895 was tested, using 0.5% human had hind limb weakness that progressed to hind limb paraple- type-O red blood cells at 22, 37, and 4ЊC. Neutralizing antibod- gia with the absence of pelvic limb flexor reflexes. Thoracic limb 201 Vol 50, No 2 Comparative Medicine April 2000

Table 1. Summary of clinical signs, histopathologic changes, and BRV isolation from affected baboons Duration of Virus Age Hospitalization Isolation ID (Sex) (mo) (days) Status Major clinical signs Major histopathologic change (brain) 10618(M) 10 4 Euthanized Ataxia, right head tilt, hemiparesis Meningoencephalitis Not done on right side 10718(M) 12 7 Died Ataxia, seizure, pelvic limb paresis Encephalitis Positive 10332(F) 22 2 Euthanized Ataxia, pelvic limb paresis Meningoencephalitis Not done 10406(F) 22 3 Euthanized Ataxia, left head tilt Encephalitis Positive 10750(M) 16 4 Euthanized Ataxia, seizure, vertical nystagmus, Meningoencephalitis Not done hemiparesis on left side 10607(M) 10 2 Euthanized Ataxia, pelvic limb paresis Meningoencephalomyelitis Positive 10895(M) 14 7 Euthanized Ataxia, weight-bearing lameness in Meningoencephalomyelitis Positive left leg, progressed to pelvic limb paresis, seizures 9578(M) 44 8 Euthanized Ataxia, head tremor Meningoencephalomyelitis Positive

Table 2. Baboon of baboon orthoreovirus (BRV) location, exposure, and incubation time Date of Residence Transfer Residence Diagnosis of Signs Possible Animal diagnosis Cage time to cage time after housed in cage (days) incubation time No. of Animals Exposed in cage ID (mo/yr) No. (days) No. (days) 104.18 104.12 (days) 104.18 104.15 104.12 102.01 10406 7/93 104.18 181 104.12 207 393 207 207–393 6 NA 2 NA 10332 4/93 104.18 181 104.12 117 298 117 117–298 6 NA 2 NA 10718 2/93 104.18 66 Hospital NA 66 NA 87 6 NA NA NA 10618 1/93 104.18 46 Hospital NA 46 NA 46 6 NA NA NA 10750 10/93 104.18 288 104.15 10 298 10 10–308 6 4 NA NA 10607 10/93 104.18 288 104.15 21 309 21 309 6 4 NA NA 10895 11/93 104.18 114 104.15 55 169 55 55–169 0 4 NA NA 9578 1/94 104.15 56 102.01 1 57 1 57 0 4 NA 0 flexor reflexes were absent unilaterally in one animal. Two ani- ciable histologic lesions that could be attributed to viral infec- mals became tetraparetic 48 hours after signs of neurologic dys- tion were not evident. function were first seen. A review of the SFBR records for the past 10 years revealed a Although it is difficult to determine the exact incubation total of 11 baboons with primary nonsuppurative encephalitis times, the cage transfer dates and the dates that clinical signs that was morphologically similar to the present outbreak. Ten were seen established a range (Table 2). The possible incuba- of eleven of the cases developed in juvenile baboons, but similar tion times, as determined from the clinical history, could vary lesions were seen in one adult baboon. from 1 to 393 days. However, baboons 10718 and 10750 were the Viral isolation and growth: The BRV 10895 induced re- first cases. They were the first to die and were kept in cage markable CPE with numerous large syncytia and central amor- 104.18 for 66 and 46 days, respectively, before clinical signs de- phous, eosinophilic inclusions in Vero cells inoculated with veloped. Baboon 9578 was not housed in cage 104.18 but devel- brain tissue(3). Virus was recovered only from brain tissue in oped clinical signs of disease within 57 days after exposure to a affected or experimentally infected baboons. Virus was not re- baboon with clinical signs. covered from CSF from affected or control baboons or from any Serum biochemical and CBC results were within normal lim- other nonbrain samples tested. The MA104 and QT6 cells were its for all animals except one, which had a slightly high white non-permissive for BRV growth; BRV grows without CPE in blood cell count (16.2 x 103 cells/␮l). All animal weights and HEp-2 cells, rhesus kidney cells, and mouse L cells, replicating temperatures were unremarkable. Culture of CSF specimens 0to titer of 102 to 103, whereas titer in Vero cells reaches 106 or did not yield bacteria. The six animals treated by use of conven- higher. Vero cells were selected for subsequent experiments. Vi- tional treatment did not respond. The progression of the disease rus was not detected in any sentinel animal, including the feral was severe and rapid, ranging from 2 to 8 days’ duration. Seven rats collected in the vicinity of the outbreak. Although throat animals were euthanized and one died. and rectal swab specimens were taken from baboons with clini- Anatomic pathology: At necropsy, gross lesions were not cal signs of disease, they were test positive only for the two ex- remarkable in the affected baboons, experimentally infected perimentally infected baboons. baboons, feral rats, or experimental mice. Bacterial culture re- Early in the study, the virus was identified as a double- sults were negative. stranded nucleic acid virus. Acridine orange caused the virus to Microscopic pathology: The eight clinical cases and the fluoresce a pale-green color. This is a classic method used (10) to two experimentally infected baboons that were evaluated histo- identify double-stranded nucleic acids because they bind only a logically had similar lesions that varied in severity, character- small amount of the dye and, therefore, stain orthochromatically. ized by lymphocytic perivascular cuffing in white and gray Nucleic acid extraction of partially purified virions was followed matter in the brain, spinal cord, and meninges. In addition to by use of sodium dodecyl sulfate-polyacrylamide gel electro- perivascular cuffing, scattered microglial nodules, areas of de- phoresis. The BRV 10895 was observed to have a 10-segment myelination, axonal degeneration, vacuolization, hemorrhage, double-stranded RNA genome. The resultant classification of and accumulation of glial cells were seen in the most severely the virus on the basis of the double-stranded, 10-segmented affected baboons (Figures 2–4). In more advanced cases with se- RNA is in the genus Orthoreovirus (3). vere necrosis, a few neutrophils were observed. Other appre- Electron microscopy: Viral particles were viewed by use of 202 Orthoreovirus Meningoencephalomyelitis in Baboons

Figure 2. Photomicrograph of a section of cerebellum from an af- Figure 3. Photomicrograph of a section of cerebrum from an affected fected baboon showing the typical vacuolization and perivascular cuff- baboon. Notice typical perivascular cuffing, areas of demyelination, ing by lymphocytes and plasma cells in the meninges and neuropil. vacuolization, and accumulation of glial cells. H&E stain; x 100. H&E stain; x 40.

TEM in thin sections prepared from infected but not control the right conditions. This complicates differential diagnosis. Vero cells. The particles were localized in the cytoplasm of in- Clearly, the virus isolated from the affected baboons had the fected cells and use of negative staining revealed nonenveloped, properties of a neuroinvasive agent, in addition to being a icosahedral virions with concentric capsids 65 to 67 nm. The double-stranded RNA virus. The neurotropism of the virus was particle morphology was consistent with members of the family indisputable because it was isolated from brain tissue of natu- Reoviridae, genus Orthoreovirus (3). rally and experimentally infected animals. The virus was iso- Serologic testing: Five serum samples from the four in- lated from rectal and throat swab specimens from fected, encephalitic animals failed to neutralize infectivity of experimentally infected baboons, which may indicate the natu- BRV 10895. Further, the virus was not neutralized by mamma- ral method of dissemination. Clinical signs of disease were not lian reovirus types 1–3 sera and did not agglutinate human seen in the two baboons 6 weeks after inoculation, possibly indi- type-O red blood cells. Of three affected baboons, only one had cating a prolonged incubation period. Histologic lesions were antibodies to Orthoreovirus type 1, whereas three of eight con- found in these experimental baboons at 6 weeks. The clinical trol baboons had antibodies against one of each of the mamma- data also indicate an incubation period of approximately 46 to lian serotypes 1–3. All sera evaluated from the affected animals 66 days. However, there was serologic evidence that this agent had reactivity when tested by ELISA and Western blot analysis was not a newly present virus in this animal colony, because that contained BRV 10895. baboon sera stored from the early 1970s reacted with virus an- The only samples, screened early in the outbreak for a pos- tigens when they were tested by use of ELISA and Western blot sible etiologic agent, that were positive other than for BRV were analysis, and file tissue specimens with morphologic lesions serum samples that reacted with mumps virus antigen with consistent with the same disease were found. These data, com- complement fixation. These were considered to be nonspecific bined with the neurotropism of the virus, indicate that patho- cross-reactions and false-positive results. logic changes induced by the virus may not always be apparent Experimental mouse study: Swiss Webster Hsl:ND4 out- in baboons, or perhaps that baboons were previously infected bred mice were susceptible to infection, with differences associ- with a variant of the current virus that induced antibodies, but ated with route of inoculation as well as inoculum used. Mice not overt disease. In any case, none of the animals had neutral- that were injected intrathecally with infected Vero cell cultures izing antibodies, which is at least one documented mechanism manifested marked clinical signs of infection within PID 7, by which virus can remain persistent within a host (14). The whereas mice that received frozen-thawed, infected brain homo- limited neurovirulence observed in the baboons without clinical genate had few or no signs of infection until PID14. Virus was signs of disease can also be related to dose of the virus, route of re-isolated from animals infected with Vero cell-passaged virus entry, and host age, species, and immune status. There are a as well as infected brain homogenate. number of viruses that initiate CNS disease in humans after At necropsy, the brain tissue from animals of neonatal groups initial replication in the respiratory tract. These include herpes- 1 and 2 was the sole source of isolated virus. Histologic lesions viruses, papovaviruses, togaviruses, and arenaviruses. Although were not apparent in the 1- or 2-week survivors, or sentinel the affected animals had evidence of herpesvirus infections mice. All other tissues appeared normal histologically. Evalua- (viz., specific antibodies), only the orthoreovirus was found in tion of necropsy specimens resulted in the discovery of virus in brain tissue. Whether the orthoreovirus acted in concert with only brain tissues from the experimentally infected mice and another agent or other factors remains unclear. Further studies virus was not recovered from any other tissue. are underway to clarify the role(s) of this agent in CNS disease and to determine its origin. Discussion The source of the virus is unknown, and reservoirs for mam- Nearly all virus groups contain members that can infect hu- malian reovirus have not been proved to exist, but it is thought man, and probably nonhuman primate nervous systems under that infected animals are carriers (8). Generally, induction of 203 Vol 50, No 2 Comparative Medicine April 2000

of encephalitis is remarkably similar, regardless of the invading virus. During the initial phase of viral infection of the CNS, mononuclear inflammatory cells are recruited and rapidly di- vide. Induction of inflammation is immunologically specific. T Cells release lymphokines, which attract macrophages; this con- stitutes most of the response. Perivascular infiltrates of lymphocytes, neuronophagia, and microglial proliferation are generally seen, as in the cases described here. B Cells may be present, but usually their differentiation into plasma cells is seen only late in the disease. The components of the inflammatory response also vary according to the stage of the disease and nature of infection; however, it should be noted that all neurotropic viruses do not induce a substantial inflammatory response in the CNS. The agent described here induces severe inflammatory response in a subset of infected baboons. Figure 4. Photomicrograph of a section of spinal cord from affected There is no indication of public health or occupational threat, baboon. Notice typical lesions of lymphocytic cuffing, axonal degeneration, demyelination, and vacuolization. H&E stain; x 100. but identification of this agent presents concern because baboon organs, tissues, and cells are used in xenograft transplantation in humans. The behavior of this virus in immunosuppressed inflammatory disease is mediated specifically by the invading hosts, particularly foreign hosts, is unknown, as is the virus’s virus. Although inflammatory infiltrates are not pathognomonic predilection for specific tissues, cells, or organs. The absence of of any particular virus agents previously discussed, they con- neutralizing antibodies despite high titer of cross-reactive se- tribute to the severity of encephalitic disease and the conven- rum antibodies raises the concern that this is a persistent virus tional appearance of the usual signs associated with viral in the baboon that is capable of causing severe CNS disease un- invasion of the CNS. der certain circumstances. For these reasons, expanded studies Virus isolation and identification from CSF or brain tissue is of this virus are underway to identify virus and virus-specific the accepted standard for diagnosing viral infection of the CNS. antibodies unequivocally, as well as host conditions conducive Candidates for isolation included picornaviruses, adenoviruses, for persistent versus acute CNS disease in the baboon and other herpesviruses, rhabdoviruses, arenaviruses, retroviruses, mammalian hosts. flaviviruses, togaviruses, filoviruses, bunyaviruses, and possibly others. Diagnosis by identification of viruses that most com- monly are associated with CNS disease requires 1 to 28 days, depending on the virus (14). The baboon could be a good model Acknowledgments The authors extend special appreciation to Marie Silva and Sherrian of reovirus persistence. It is possible that viral infection is not McAnn for anatomic pathology work. This work was supported by limited to the CNS, and virus can be isolated from other sites. NIH grant P40 RR05163. Enteroviruses (family Picornavirus) may be isolated from serum, or throat or rectal swab specimens (15). Virus could be isolated only from brain tissue of naturally infected baboons and References throat and rectal swab specimens were virus positive only for 1. Brizzee, K. R. 1972. Diseases of the nervous system. p.542–638. experimentally infected baboons. Acridine staining suggested In R.N.T.-W. Fiennes (ed.), Pathology of simian primates, Part 1: General pathology. 1st ed. Karger, Basel. an RNA virus and purification of nucleic acids confirmed the 2. Kim, C. S., F. H. Kriewaldt, N. Hagino, et al. 1970. Subacute virus contained double-stranded RNA comprising 10 segments, sclerosing panencephalitis-like syndrome in the adult baboon several of which co-migrate together (3). (Papio sp.). J. Am. Vet. Med. Assoc. 157:730–735. 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