1983 pathogenesis of coronavirus sd in mice

Pathogenesis of Coronavirus SD in MiceI. Prominent Demyelination in the Absenceof Infectious Virus ProductionPaul M. Mendelman, MD; Lynn D. Jankovsky, DVM, PhD; Ronald S. Murray, MD;Patricia Licari; Bonnie DeVald; John C. Gerdes, PhD; Jack S. Burks, MD

\s=b\Following intracerebral inoculationof 3- to 4-week-old C57 B 16/J mice withcoronavirus SD, 23% exhibited neurologicsigns within the first week. However, only6% died. Within the first week after inoc-ulation (AI), we noted a panencephalitis.Prominent demyelination detected in thespinal cord on day 6 continued throughday 29 AI. Demyelinated lesions in thespinal cord were either subpial with fewinflammatory cells except for macro-

phages or perivascular with prominentaccumulation of lymphocytes, plasmacells, and macrophages. Beginning on

day 6 AI, IgG was detected in the lesions.Although an infectious virus was detect-able in the CNS only through day 12 AI,viral antigen expression continuedthrough day 24. We concluded that coro-navirus SD persists in a nonrecoverableform throughout the initial phase of demy-elination, day 6 to day 24 AI.

(Arch Neurot 1983;40:493-498)

XTuman demyelinating diseases suchas progressive multifocal leu-

koencephalopathy, subacute sclero-sing panencephalitis, and postinfec-tious or postvaccinal encephalomyeli-tis are known to be initiated by viral

agents.1 A viral cause has also beensuspected for one of the most commonhuman demyelinating diseases, multi-ple sclerosis (MS).1-2 We previouslyreported the isolation of two corona-viruses while working with CNS tis-sue of two patients with MS.3 Both iso-lates were closely related serologicallyto the human coronavirus OC43, aswell as to the nonneurotrophic murinecoronavirus A59.4 Following intra-cerebral inoculation, most murinecoronaviruses produce dramatic,acute hepatitis.5 However, the neuro-

trophic murine coronavirus strainJHM produces a chronic demyelin-ating, remyelinating disease followingintracerebral (IC) inoculation of miceand rats.612 It has also been reportedthat the three togaviruses—SemlikiForest virus,13 Ross River virus, andVenezuelan equine encephalomyelitisvirus15—can cause demyelination inmice. Since coronavirus SD is antigen-ically distant from strain JHM, it wasinteresting to determine its pathogen-esis in mice. We describe demyelin-ation produced by coronavirus SD iso-late following IC inoculation of wean-ling mice.

MATERIALS AND METHODSWe obtained 3- to 4-week old, male,

specific pathogen-free C57 B16/J mice. Ahanimals were maintained under specificpathogen-free conditions throughout thestudy. Uninoculated control mice, housedwith the study animals, served as monitorsof these conditions.Neutralizing antibody was detected by

the 50% plaque neutralization assay ondelayed brain tumor (DBT) cells." Serumwas pooled at the time of collection, inacti-vated at 56 °C for 30 minutes, stored at —70°C, and thawed at the time of assay. Serumfrom control mice, preimmune mice, and

fetal calves was run for negative controls.All serum was run in duplicate. A total of110 infected mice at 24 time points (aver-age, 4.6 mice) and 32 control mice at 17time points (average, 1.9 mice) were tested.Preimmune serum titers to coronavirus SDwere undetectable.

We inoculated 388 mice IC with 10,000 to100,000 plaque-forming units (0.03 mL) ofplaque-purified SD virus. An additional137 mice were inoculated IC with unin-fected DBT cell extract. All inoculationswere performed using ether anesthesia.For viral isolation studies, two or three

mice were killed at each time point. Bloodand tissue were collected while the micewere under ether anesthesia. Whole bloodand tissues were immediately ground witha mortar and pestle on ice and suspendedto 10% in Hanks' balanced salt solutionsupplemented with penicillin and strepto-mycin. Suspensions were clarified by cen-trifugation at 12,800 g for three minutesand stored at -70 °C. Supernatant viruswas detected by plaque assay on DBTcells.4 Brain and spinal cord tissue wasminced and co-cultivated on monolayers ofDBT cells or cultured directly for later cellfusion with 3T3 (17C1-1) cells."For in situ viral antigen studies, brains

and spinal cords were immediately snapfrozen in a dry ice-alcohol bath and storedat —70 °C. Four-micrometer cryostat sec-tions were fixed in acetone for ten minutesat -20 °C and air dried. The sections weresoaked for ten minutes in phosphate-buf-fered saline (PBS) solution with a pH of7.0. Preimmune or viral specific antisera4were applied (50 pL). Siliconized cover-

glasses were added and the primary anti-sera allowed to react for 30 minutes atroom temperature in a humidified cham-ber. Coverglasses were then gently soakedoff. Following two ten-minute rinses inPBS, 50 uL of either fluorescein-con-jugated sheep anti-guinea pig IgG, Staphy-lococcus aureus protein A labeled withiodine 125 (50,000 counts per minute persection), or rabbit anti-guinea pig IgG 12'I

Accepted for publication Dec 29, 1982.From the Division of Infectious Disease,

Department of Pediatrics, Childrens OrthopedicHospital and Medical Center, School of Medicine,University of Washington, Seattle (Dr Mendel-man); Departments of Pathology (Dr Jankov-sky), Neurology (Drs Murray, Gerdes, andBurks), and Immunology (Drs Gerdes andBurks), Rocky Mountain Multiple Sclerosis Cen-ter, University of Colorado Health Sciences Cen-ter, School of Medicine; and Veterans Adminis-tration Medical Center (Mss Licari and DeValdand Drs Gerdes and Burks), Denver.Reprint requests to University of Colorado

Health Sciences Center, Box B181, 4200 E NinthAve, Denver, CO 80262 (Dr Burks).

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Days AI

Fig 1.—Infectious virus detected in 10%homogenates of brain (squares), spinal cord(triangles), and spleen (circles) by plaqueassay on delayed brain tumor cells afterintracerebral inoculation of SD virus in wean-ling C57 B16/J mice. AI indicates after inocu-lation; PFU, plaque-forming units.

(50,000 counts per minute per section) wereadded. Coverglasses were placed on thesections, and labeling continued for 30minutes at room temperature. Protein A125I had a specific activity of 60 mCi/mg.Rabbit anti-guinea pig IgG was iodi-

nated using an iodogenic method" and freeiodine was removed using desalting col-umns as described by Christopherson etal." Average specific activity of iodinatedrabbit anti-guinea pig IgG was from 1million to 10 million counts per minute permicrogram of protein. For autoradio-graphie techniques, unreacted iodine wasremoved by two ten-minute washes in PBS,followed by an overnight rinse at 4 °C in 4L of PBS. Sections were then dehydratedwith two ten-minute washes of 70% alco-hol and two ten-minute washes of 95%alcohol, air dried, and stored at 4 °C insealed slide boxes containing anhydrouscalcium sulfate. For autoradiography, thesections were soaked for ten minutes inPBS and dipped at 42 °C in photographicemulsion (NTB-2) diluted 1:1 in distilledwater containing 2% glycerol. Our autora-diographic antigen detection methods weredescribed in detail elsewhere" and weresimilar to those of Moar et al.20 Following aseven-day exposure at 4 °C, slides weredeveloped, rinsed, and fixed. Tissue sec-tions were stained using a modified hema-toxylin-eosin procedure as described byBaserga and Malamud.21For histopathologic studies, anesthe-

tized mice underwent perfusion throughthe left ventricle of the heart with bufferedformaldehyde solution until blanching ofthe liver occurred. Representative tissuesfrom all major organ systems wereremoved and stored in excess perfusate atroom temperature. They were then em-bedded in paraffin, sectioned at 5 urn, andstained with hematoxylin-eosin for routinescreening. Serial sections of the brain andspinal cord were also stained with Luxolfast blue PAS-hematoxylin, phosphotungs-tic acid-hematoxylin, and the Bodianmethod.

Mice processed for electron microscopy

Fig 2.—Autoradiographic detection of viral antigen in focal areas of increased cellularity intemporal cortex of infected mouse four days after inoculation (AI). Top left, Light microscopy(original magnification X410). Top right, Darkfield microscopy (original magnification X410).Bottom, Background level of exposed silver grains observed by light microscopy (left) anddarkfield microscopy (right). This section was reacted with preimmune guinea pig antiserum andrabbit anti-guinea pig IgG labeled with iodine 125, then exposed for seven days prior todevelopment. Similar background levels were obtained on uninfected tissue reacted with guineapig antiviral antisera and rabbit anti-guinea pig IgG l26l or sections of brain or spinal cord reactedwith protein A '"I (original magnification X410).

were first heparinized with 10 units. Eachanimal was perfused with 5 mL of cacody-late buffer (pH, 7.3), followed by 30 mL ofhalf-strength Karnovsky's fixative at roomtemperature. Selected tissues were re-moved, diced to 1-mm cubes, and stored inexcess perfusate overnight at 4 °C. Tissueblocks were postfixed in 1% osmiumtetroxide and 1.5% potassium ferricya-nide22 for one hour. Dehydrated tissueswere subsequently embedded in epoxy res-in. Thin sections were stained with uranylacetate-lead citrate.

RESULTS

Clinical signs were first observedbetween days 4 and 9 AI, during which91 (23.4%) of 388 infected animalsdisplayed either extreme hyperexcit-ability or hind limb paresis. Twenty-

five (6.4% ) of the infected mice exhib-iting symptoms died during this firststage. Subsequently, all mice becamefree of clinical signs, except for con-tinued growth retardation in a fewrecovered animals, until day 30 AI.From day 30 to day 60 AI, 26 (26.8% )of 97 infected mice had clinical evi-dence of hind limb anesthesia and/orparesis. Hind limb anesthesia wasshown by a lack of response to lighttouch or pinprick sensation, whereasparesis was noted when animals wereweak and dragged their hind limbs. Ofthese animals, 12 eventually becamemoribund and died. Only six had evi-dence of previous disease betweendays 4 and 9 AI. Neutralizing anti-body to coronavirus SD was detect-


Histopathology Following Intracerebral Inoculation of Coronavirus SD*

Days After Inoculation

_t-5_6-10_11-15 16-20 21-25 26-30 31-60 61-90 91-120No. of mice studied (No. ofcontrols)_22(10) 20(10) 16(8)_6(3) 3(1) 6(3) 10(5) 2(1)_6(2)

Histopatologic evidence, No. (%)MeningitisCerebral_20(90.1) 15(75)_2(12.5) 0(0) 0(0) 0(0)_0 (0) 0 (0)_0(0)Spinal cord_22(100) 12(60) 0(0)_0 (0) 0 (0) 0 (0)_0 (0) 0 (0)_0(0)

Encephalitis_18(82) 20(100) 14(87.5) 1(17) 2(33) 0(0)_0 (0) 0 (0)_0(0)Myelitis_16 (72) 17 (85)_7 (44)_4 (67) 1 (33) 2 (33) 0 (0) 0 (0)_0(0)Demyelinationt_0(0)_6(30)_9(56)_4 (67) 1 (33) 2 (33) 0 (0) 0 (0)_0(0)Hydrocephalus_0(0)_0(0)_5(31)_3 (50) 2 (67) 3 (50) 4 (40) 1 (50)_0(0)

'Numbers represent the number ol animals lor which the given lesion was observed.tThe (requency of demyelination is that observed in longitudinal sections of formaldehyde solution-fixed, paraffin-embedded spinal cord. Extensive examination of

1-^m cross sections of epoxy resin-embedded spinal cord showed demyelination in two ol lour mice on day 6 after inoculation, four of four on day 8, four of four on day12, four of four on day 28, and two of five on day 90.

able beginning on day 4 AI. The 50%plaque neutralizing titer increased toa mean of 1:2,560 by day 9 AI, remain-ing at this level for the duration of thestudy. Neutralizing antibody was nev-er detected in control animals.Infectious virus was recoverable in

high titer from the brain and spinalcord, and in considerably reduced titerfrom the spleen and liver for the firstfour days AI (Fig 1). Virus could notbe isolated at any time from the blood,heart, lung, thymus, kidney, or duode-num. Infectious virus was not de-tected in the liver by day 5, the spleenby day 6, and the brain and spinalcord by day 7. Virus was detectable byco-cultivation of minced brain tissueon either DBT or 3T3 (17C1-1) cells aslate as day 12 AI, after which allmethods of isolation, tissue homoge-nate, co-cultivation, and cell fusionproved unsuccessful. An extensivesearch for viral particles by electronmicroscopy revealed little except anoccasional viral particle in necroticcell debris of the temporal cortex on

days 4 through 6 AI.Evidence of viral antigenic expres-

sion was seen in sections of brains andspinal cords by indirect immunofluo-rescence for the first ten days AI.Until day 24 AI, virus could bedetected using anti-guinea pig IgG 125Iand autoradiography. Viral antigenwas widely distributed in the menin-ges and predominated in the whiteand gray matter of the brain andspinal cord. However, distributiontended to be heaviest and persistedthe longest in temporal cortex graymatter of the brain (Fig 2) and inspinal cord white matter. Using pro-tein A ,25I, IgG was found to infiltratebrain and spinal cord lesions begin-ning on day 6 AI. Serial sections ofspinal cord lesions showed both IgGand viral antigen expression in identi-cal lesions. Prior to day 6, viral anti-

gen but no IgG was found in areas ofinflammation.

To determine if viral antigenexpression represented persistence ofinfectious virus or expression of alatent or defective virus, the frozentissue blocks adjacent to antigen-posi-tive areas were thawed, minced, andco-cultivated with 17C1-1 cells. Withthis method, infectious virus could bedetected through day 12 AI, but it wasnot recovered from any tissue blocksbetween days 12 and 24 AI.

Histopathology of the Brain

Histopathologic evidence of CNSinvolvement was encountered in 95%of infected mice examined during thefirst 29 days AI (Table). A light cellu-lar infiltrate predominantly com-

posed of neutrophils and occasionalmultinucleate syncytial cells was seenin the cerebral meninges and near theinoculation wound in the left cerebralhemisphere on day 1 AI. Both thehippocampus and temporal cortexwere prominently involved by day 2AI. Neuronal necrosis was striking,especially in the temporal cortex, andboth areas were heavily infiltrated byneutrophils. Acute panencephalitisdeveloped in animals examined on day4 AI and persisted through day 8. Byday 9, the inflammatory cell responsein the brain was limited to the tempo-ral cortex and its overlying meninges.The nature of the response hadchanged in that mononuclear cellsnow predominated and neuroglialnodules were evident. Portions of thetemporal lobes, which on day 7 AIwere acutely necrotic, underwent liq-uefaction on days 9 through 13 AI.Mineral deposits were occasionallyobserved in the temporal cortexbeginning on day 12 AI. The destruc-tive changes in the temporal cortexled to the development of compensato-ry hydrocephalus. During this early

period, myelin destruction was noted,primarily in the temporal cortex.

Histopathology of the Spinal Cord

Although inflammatory lesionswere found in the spinal cord and thesurrounding meninges from days 1through 17 AI, they lacked the inten-sity of the cerebral lesions andremained focal. Demyelination was astriking feature that was firstobserved on day 6 AI and persisteduntil day 29 AI. Initial lesions weresmall and subpial. By day 17 AI, largeareas of demyelination extended overseveral vertebral segments (Fig 3,top). Subpial edematous lesions werenot usually associated with inflamma-tory cells other than macrophages. Byelectron microscopy, myelin strippingby these macrophages was noted (Fig4, bottom right). Other lesions, partic-ularly prominent at day 12 AI, tendedto be larger and deeper and were

adjacent to prominently cuffed ves-sels (Fig 3, bottom). The perivascularcuffs consisted of lymphocytes, mac-

rophages, and plasma cells (Fig 4, topand bottom left). In areas of bothtypes of demyelination, axon preser-vation was shown by both light andelectron microscopy (Fig 3, top right,and Fig 4, bottom ¡eft).

COMMENT

Following IC inoculation of coro-navirus SD into weanling mice, prom-inent foci of demyelination weredetected in the spinal cord. The devel-opment of demyelinating lesions fol-lowing infection was especially inter-esting since coronavirus SD was iso-lated while we were working with MSautopsy brain material,3 and demye-lination occurred in the absence ofinfectious virus production.3Although the cause of MS is

unknown, considerable evidence sug-gests viral involvement in its patho-


Fig 3.—

Demyelination in spinal cord following intracerebral inoculation of coronavirus SD inweanling mice. Top left, Longitudinal section of diffuse spinal cord demyelination 13 days afterinoculation (AI) (hematoxylin-eosin, original magnification X40). Top right, Longitudinal sectionof focal spinal cord demyelination with numerous gitter cells and well-preserved axons 20 daysAI (Bodian method, X40). Bottom left, Cross section showing perivascular demyelination andmononuclear cell cuff in spinal cord white matter 12 days AI. Embedded in epoxy resin (toluidineblue, original magnification X164). Bottom right, Mononuclear cell cuff and adjacent demyeli-nation (X256).

genesis.1-2 Coronaviruses have beenspecifically implicated as a possiblecause since (1) Tanaka et al23 foundcoronaviruslike particles by electronmicroscopy in the brain tissue of onepatient with MS; (2) coronavirusesJHM and SD are capable of causingdemyelinating disease in mice612; (3)coronaviruses were isolated whileworking with fresh autopsy materialfrom two patients3; (4) sérologie inves-tigations showed elevated CSF coro-navirus antibody levels in patientswith MS2425; and (5) coronaviruseswere isolated from the CSF ofpatients suffering from an acuteencephalitic syndrome.26 While a rolefor coronavirus SD in the pathogène-

sis of MS has yet to be established, a

comparison of SD-induced demyelin-ation in mice with MS and othervirus-induced demyelination in ani-mal models is appropriate.The characteristic lesions of MS are

focal areas of demyelination in whichaxons appear relatively well pre-served and oligodendroglia are de-stroyed.2' Acute lesions often appearedematous and may be associatedwith perivascular accumulations oflymphocytes, plasma cells, and macro-

phages. Lipid-filled macrophages are

numerous, especially at the marginsof acute lesions. Although the distri-bution of lesions varies widely withinthe CNS, they are typically located in

subpial, subependymal, and/or peri-vascular areas.27

One theory of the cause and patho-genesis of MS postulates that itresults from an immunopathologicprocess in genetically predisposedpersons in response to a viral infec-tion. Evidence of an immunopatholog-ic process consists of observation ofperivascular lymphoid cells in acutelesions,27 decreased numbers of circu-lating suppressor T lymphocytes dur-ing exacerbations,28 accumulation ofimmunoglobulin associated with de-myelinated lesions,29 and the presenceof elevated oligoclonal IgG in the CSFof patients with MS.25Multiple sclerosis is a disease

unique to humans. In fact, naturallyoccurring demyelinating diseases ofanimals are rare. Animal models ofdemyelination depend on the viralstrain involved, the age and strain ofanimal infected, and the inoculationroute.612'31 Two distinct mechanisms ofdemyelination have been demon-strated. One involves a direct viralcytolysis of oligodendroglia, the sec-ond an immunopathologic process.Murine hepatitis viral infection

(strain JHM) is the main example ofdemyelination resulting from viralcytolysis of oligodendroglia. Charac-teristically, lesions appear as random-ly distributed foci of demyelinationlacking inflammatory cells other thanlipid-filled macrophages. Immuno-globulin G is absent from theselesions; however, viral antigen isabundant. By electron microscopy,viral particles are recognized in oli-godendroglia.9 Immunosuppressiondoes not prevent demyelination."Intracerebral inoculation of mice

with the DA strain of Theiler's virus,32selected temperature-sensitive mu-tants of vesicular stomatitis virus,33 or

Chandipura vesiculovirus34 results ina prominent mononuclear inflamma-tory reaction adjacent to foci of demy-elination in the spinal cord. The cellbodies of oligodendroglia are unaf-fected. Immunosuppression reducesthe inflammatory reaction and demy-elination.32 Viral particles are usuallynot observed by electron microscopy.The primary demyelination follow-

ing inoculation of coronavirus SD ismorphologically similar to that ofacute lesions in MS. The demyeli-nating lesions are subpial or perivas-cular. Mononuclear and prominentIgG infiltration occur concomitantlywith demyelination. The pathogenesisobserved indicates that both oligoden-droglia destruction and subsequentimmunopathologic reactions may oc-cur. Virologie studies during the peri-


Fig 4.—Electron micrograph showing perivascular cell infiltration and phagocytic myelinstripping. Top left, Mononuclear cells migrating into perivascular area of demyelination 12 daysafter inoculation (AI), including plasma cell (lower right corner) and macrophage containing lipidvesicles and myelin debris (X4.205). Top right, Plasma cells and demyelinated axon in adjacentarea (X4.205). Bottom left, Area of demyelination 12 days AI with numerous denuded axons(X3.472). Bottom right, Phagocytic cell actively stripping myelin from axon on day 8 AI(X3.413).


od of demyelination indicate that thevirus persists in a form not readilyrecoverable. However, demyelinationis associated with a low level of viralantigenic expression. Despite exten-sive electron microscopy, viral parti-cles have not been observed in brainor spinal cord cells following corona-virus SD infection.

Regarding the possible role of coro-navirus SD in MS, the observation

that this virus persists in a nonrecov-erable state during the period ofmurine demyelination suggests thatvirus isolation may not be the bestapproach for demonstrating viralinformation in human tissue. Stan-dard antigen detection methods suchas immunofluorescence also may lacksensitivity for viral antigen expres-sion. Therefore, the definitive methodfor establishing a coronavirus etiolo-

gy for MS will require the direct dem-onstration of viral antigen or nucleicacid in MS tissue. In addition, tissuefrom autopsies in controls should benegative for viral genome.

This research was supported by the KrocFoundation and Veterans Administration grant1169.

Karen Michelsen and Michèle Reed helped inmanuscript preparation. Irene McNally providedtechnical assistance.

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1983 pathogenesis of coronavirus sd in mice

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