INFECTION AND IMMUNITY, Feb. 1976, p. 590-599Copyright © 1976 American Society for Microbiology

Vol. 13, No. 2Printed in U.SA.

Newcastle Disease as a Model for Paramyxovirus-InducedNeurological Syndromes: Pathogenesis of the RespiratoryDisease and Preliminary Characterization of the Ensuing

EncephalitisJACK G. STEVENS,* ROBERT M. NAKAMURA,' MARGERY L. COOK,

AND SHARON P. WILCZYNSKIReed Neurological Research Center and Department of Microbiology and Immunology,* UCLA School of

Medicine, Los Angeles, California 90024

Received for publication 22 July 1975

Various clinical, virological, immunological, and morphological aspects ofvelogenic Newcastle disease were defined in chickens inoculated by naturalroutes with the Missouri-(H) Len 1950 strain. The disease initially appeared as asevere pneumonitis from which most birds recovered. Several days later, manyof these birds developed severe encephalitic signs, largely referable to inflamma-tory changes in the cerebellum. During the pneumonic stage, virus was easilyisolated in relatively high titers from the brains of all chickens, and viralproducts were easily detected in Purkinje neurons. However, when the encepha-litis developed, virus was isolated irregularly and in low titers from brains, andmorphological evidence for the presence of viral products could no longer beobtained. The encephalitic disease is discussed in relation to encephalitic syn-dromes induced by other neurotrophic viruses.

An etiologic role for paramyxoviruses in en-cephalitic syndromes of diverse types is nowwell appreciated. In man, the most commonagents involved are mumps virus, which usu-ally induces a self-limiting meningitis (onlyrarely damaging the brain parenchyma [10,11]), and measles virus, which is responsible formore serious syndromes including postinfec-tious encephalitis (11, 13) and subacute scleros-ing panencephalitis (cf. review by Weiner etal. [15]). In addition, over many years, evidencehas accumulated which strongly suggests thatthere is a causal relationship between measlesvirus and multiple sclerosis (3). From theseconsiderations, it is clear that a spectrum ofneurological diseases ranging from benignmeningitis to severe and fatal illnesses are in-duced by these agents. Classically, such ill-nesses follow by some days or longer a morecommon form of disease induced by the agent inone or more other organ systems.The pathogenesis of these diseases is ob-

viously complex and their importance justifiesthe extensive study of diverse experimentalsystems. To date, most of these systems haveinvolved the human agents given by unnaturalroutes in unnatural hosts, usually intracere-

' Present address: Department of Pathology, Scripps Clinic,Medical Center and Hospital, La Jolla, Calif. 92037.

brally to young hamsters (4, 9, 14). VelogenicNewcastle disease in the chicken is a naturalmodel that has not been studied in this context.Therefore, we chose to define the pathogenesisof this disease in some detail and to concentrateultimately on several aspects of the centralnervous system (CNS) disease. In this commu-nication, we describe the pathogenesis of theacute pneumonitis and more generalized infec-tion and present an initial characterization ofthe encephalitis that follows some days afterresolution of the primary illness.

MATERIALS AND METHODSEmbryonated hen's eggs and chickens. Hatching

eggs (type 2 SPF) were obtained from Heisdorf andNelson Farms, Redmond, Wash., and hatched in ourlaboratories. To obviate accidental infection, thebirds were reared in a building separated from thelaboratory by personnel who were not allowed tocontact infected birds.

Virus. The virus employed was Missouri-(H) Len1950, a velogenic strain kindly supplied by the New-castle Disease Virus (NDV) Repository, Departmentof Veterinary Science, University of Wisconsin,Madison. Virus stocks were prepared by standardprocedures in the choriallantoic sac of SPF eggs.These stocks possessed titers ofabout 5 x 10 plaque-forming units (PFU)/ml and were stored at -70 C.Viral assays were performed on secondary chickembryo fibroblast monolayer cultures maintained in

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NEWCASTLE DISEASE 591

minimal essential Eagle medium (MEM) and 10%newborn calf serum in plastic flasks (Falcon Plas-tics, 25-cm growth area). After adsorption of 0.2 mlof a virus dilution for 1 h, the cells were overlayedwith minimal essential Eagle medium plus 10%tryptose phosphate broth, 10% newborn calf serum,and 0.6% Noble agar (Difco). After 2 days of incuba-tion at 39 C, the overlay was poured off, the mono-layer was stained with crystal violet, and plaqueswere enumerated.

Inoculation of chickens. Chicks, 2 to 4 weeks old,were used throughout the experiments. The virusstock was diluted to 10,000 PFU/ml, and 1 drop (froma 26-gauge needle) was placed on each eye and eachexternal nasal opening.

Processing of tissues. (i) Viral titrations. At ap-propriate intervals, similar tissues were removedfrom each of four birds, pooled and ground in TenBroeck homogenizers as 10% (wt/wt) suspensions.The choroid plexi were prepared as 0.1% suspen-sions, and whole blood was allowed to clot andground in a mortar with sand also as a 10% (wt/wt)suspension. For assays of blood components, thecellular components from heparinized blood wereseparated and assayed after homogenization (10%suspensions), and plasma was assayed directly. Allsuspensions were centrifuged for 5 min at 3,000 x gin a Sorvall RC-2B refrigerated centrifuge, and thesupernatant fluid was used for viral assays on sec-ondary chick embryo fibroblasts as described above.

(ii) Standard histopathology. Appropriate tissueswere fixed in neutral buffered Formalin, sectioned,and stained with hematoxylin and eosin.

(iii) Immunofluorescent techniques. For maxi-mum sensitivity, the indirect immunofluorescentmethod was used to detect NDV antigens in frozensections of appropriate tissues. Sections of tissuewere cut in a cryostat at 6 gm and fixed in acetonebefore staining. A chicken anti-NDV serum wasused as the primary reagent, and the secondaryreaction employed rabbit-derived fluorescein-conju-gated anti-chicken immunoglobulins. The chickenserum was prepared from birds that had recoveredfrom experimentally induced Newcastle disease andwere subsequently hyperimmunized with virus inFreund adjuvant. At a 1:100,000 dilution, the serumcollected from these birds neutralized about 25% ofthe added PFU of virus after 1 h at 37 C. Appropri-ate control tissues indicated that the technique wasspecific for viral antigens in all but lymphoid tissuesand cells (which normally possess levels of chickenimmunoglobulins that are stained by these meth-ods).

Electron microscopy. Since it was necessary toperform additional tests on the same tissues to beexamined ultrastructurally, infected birds were notperfused with fixative. Only tissues that were foundto contain significant amounts of viral-specific anti-gens (as shown by immunofluorescent methods)were chosen for study by electron microscopy. Birdswere sacrificed by decapitation and a portion of theappropriate tissue was placed in 1.5% glutaralde-hyde in 0.1 M cacodylate buffer and immediatelychopped into small pieces. These were stored infresh fixative until the extent of viral involvement

of the tissue was evaluated. At that time, the blockswere prepared for electron microscopy as was previ-ously described (6).

Detection of serum-neutralizing antibodies.Chickens were bled from the heart before they weresacrificed for collection of other tissues, and theserum obtained was frozen at -70 C. When all sam-ples had been collected, each sample was combinedwith the sera from other birds sacrificed on the sameday and the pool was assayed by standard methodsfor viral-neutralizing antibody.

RESULTS

Clinical course of infection. The accumu-lated results of several experiments (a total ofmore than 300 birds inoculated) serve as a basisfor description of the clinical illness. By day 4after infection, all chicks inoculated on the na-sal and ocular mucosa (as described in the pre-vious section) began to develop severe respira-tory signs with difficult breathing, mouthbreathing, and gasping. In addition, all showedobvious generalized signs such as droopingwings, ruffled feathers, and anorexia. Over thenext 3 days, one-third to one-half became som-nolent, a few developed head tremors or clonicconvulsions, and most of these birds died. Thus,in individual experiments, all birds developedrespiratory signs and one-fourth to one-halfdied by day 7. At about 7 days, the respiratoryand generalized disease began to stabilize in theremainder of the birds and a resolution of signsbegan. However, beginning at about 11 or 12days after infection, about one-third of those re-maining developed severe signs in the CNS.Most of these birds presented with torticollisand ataxia, and some had unilateral or bilat-eral paralysis of wings or legs. Although someof these birds died rapidly, or with a more pro-longed downward course (3 to 4 weeks), theconditions of most stabilized, and a few even-tually recovered.

Viral assays of various tissues. To establishthe general features of viral pathogenesis inthese birds, the birds were killed and pertinenttissues were homogenized and assayed for virusat intervals after infection. In these experi-ments, only living birds were assayed, and theywere randomized as to degree of illness. Inassays from day 12 on, most groups had one totwo birds with severe CNS signs included. Theresults of such an initial experiment are pre-sented in Fig. 1. Here, after replication in thetissues at the site of entry, virus appeared by 3days in the trachea, lungs, and blood, and byday 5, the brain was infected. This experimentalso suggests that peak titers of virus werereached in most tissues by day 5. By day 7,virus became undetectable in all tissues except

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Most of the virus was leukocyte associated atthe time of peak titers (day 4) and disappeared

6 from all tissues, again by day 7.-\11t1 (ii) Lymphoid organs. Viral assays of the

1l bursa of Fabricius, thymus, and spleen are pre-sented in Fig. 3. In general, the findings mirror

I 1I those described for leukocytes from peripheral10 blood, although it appears that less virus is as-

sociated with the thymus, and disappears fromIXA //V

this organ earlier than from the other two tis-¶BRsFAINV sues studied.

c o1 (iii) CNS. Selected portions of the CNS were

I1l1\ \ separated and assayed. As shown in Fig. 4,BLOOD1lcerebella and choroid plexi contained the most

virus, and by day 4 the agent could be detected

0a / 1, ,| " \zlin all parts of the brain. As with other tissues,LU0G4/|11\\'1the virus could not be detected in the brain by

TRACHEA day 9./,1\ .< Viral assays of brains from individual birds.

The brains from birds sacrificed during the pe-o2_ l \L\ riod of acute pneumonia (5 days after infection),

]t_1 X -t_-- _ - -:4 or on the day that CNS disease developed (11 toCONJUNCTIVAL NASAL MUCOSA

101- _ _3 5 7 9 12 15

TIME (days) \

FIG. 1. Amount ofNDVpresent in various tissues 105- WHITE BLOOD CELLSassayed at intervals after intranasal and conjuncti- <val inoculation ofchickens. Similar tissues from four -'chickens were pooled and assayed at each interval _a|noted. Details concerning methods are given in the /text. The level of sensitivity for these assays is 50 104-

0.PFUIg oftissue.,, - t '-JI

lung, where it disappeared by day 9. Althoughthe tissues for assay were not taken at daily Iintervals, this experiment puts the virological , / /aspects of this disease into a general time -frame. Of particular significance to subsequent E /considerations is the fact that virus was easily '\detectable in all tissues examined at 5 daysafter infection but could not be recovered fromany tissue by day 9. -ED BLOOD CELLS

D)etailed virological studies of selected tis-sues. Since we were more interested in thoseaspects of the disease associated with the cen- .0tral nervous and hematological systems, a 5_ .PLASMA.more detailed virological study of these tissues I -was completed. In all cases, chickens were in- 3 5 7 9 11 13 15fected and tissues were processed as described TIME (days)earlier. FIG. 2. Amount ofNDV present in hematological

(i) Peripheral blood. When the three main components assayed at intervals after intranasal andcomponents of peripheral blood were separated conjunctival inoculation of chickens. Similar tissuesneass f .

(and fluids from four chickens were pooled and as-

and assayed for virus (Fig. 2, it was found that sayed at each interval noted. Further details concern-

a considerable amount of virus was associated ing methods are given in the text. The level of sensi-with leukocytes and erythrocytes by day 3, but tivity for these assays is 50 PFUIg for blood cells andnone was detectable in plasma until day 4. 5 PFU/g for plasma.

INFECT. IMMUN.592 STEVENS ET AL.

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NEWCASTLE DISEASE

107-

106

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TIME (days)

FIG. 3. Amount ofNDV present in lymphoid tis-sues assayed at intervals after intranasal and con-junctival inoculation of chickens. Similar tissuesfrom four chickens were pooled and assayed at eachinterval noted. Further details concerning methodsare given in the text. The level of sensitivity for theseassays is 50 PFUIg of tissue.

17 days after infection), were assayed individ-ually for virus. In these more sensitive andprecise assays, virus could be detected in smallquantities (50 to 103 PFU/g of tissue; average, 2x 102 PFU/g) in only one-half of the brains(6 of 12) of birds sacrificed on the day that CNSsigns developed. On the other hand, all birdsstudied (12 of 12) that were killed on day 5 ofinfection had virus in the brain and in signifi-cant amounts (2 x 102 to 4.8 x 101 PFU/g oftisue; average, 1.6 x 104). This latter group wasselected so that more than one-half would havesurvived the acute disease.Appearance of neutralizing antibody. Neu-

tralizing antibody become detectable by days 4to 5 after infection (neutralization constant [K]at 37 C [1] = 0.2 ml/min at 4 days and 0.4 ml/min at 5 days) and had apparently not reacheda plateau by day 15 when the experiment wasterminated (K = 7.3 ml/min).

Pathological effects. (i) Light and fluores-

cence microscopy. In this and succeeding sec-tions, emphasis will be placed on the morpho-logical changes that occurred in the CNS, al-though other pertinent tissues will be presentedin less detail. In all cases, only prominentchanges will be discussed. Study of the respira-tory system was limited to a gross and histo-pathological description of the pneumonia.Grossly, the lungs were enlarged, hyperemic,wet, and totally involved. Microscopically, asevere hemorrhagic pneumonia was noted,with a fibrinous exudate containing both eryth-rocytes and leukocytes in the parabronchi andlarger airways.

Gross appearance of the lymphoid tissueswas generally unremarkable, except forsplenomegaly. Microscopically, by day 5 andthrough day 9 the spleens and thymusesshowed focal vacuolation and destruction ofcells in cortical areas and germinal centers.Lymphoid follicles of the bursa demonstratedsimilar effects on lymphocytes, with the medul-lary area most markedly involved. By day 12,these organs showed no striking alterations ex-cept for some residual splenic congestion.

(ii) CNS. In the CNS, lesions became promi-nent on days 6 and 7. Vascular changes, most

v-r-i commonly a perivascular accumulation of13 15 round cells in the white matter of cerebellar

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FIG. 4. Amount of NDV present in various por-tions of the brain assayed at intervals after intran-asal and conjunctival inoculation of chickens. Simi-lar tissues from four chickens were pooled and as-sayed at each interval noted. Further details concern-ing methods are given in the text. The level of sensi-tivity for the assays is 5 x 10:' PFUIg for choroidplexus and 50 PFU/g for all other tissues.

VOL. 13, 1976 593

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FIG. 5. Photomicrograph ot cerebellar white matter from a chicken processed 6 days after the bird wasinoculated with NDV. Two uessels demonstrating pernvascular inflammatory changes are prominent ( x280).

FIG. 6. Photomicrograph of cerebellar cortex from a chicken processed 11 days after the bird was inocu-lated with NDV. A large glial nodule is present in the molecular layer, and the Purkinje cells beneath thisnodule have disappeared (x280).

folia and nuclei, were noted in all birds (Fig. 5).The meninges of the cerebellum demonstratedan increased number of mononuclear cells, andall areas of the brain were edematous and con-

gested. By day 9, there was disappearance ofPurkinje cells and focal areas of glial and mono-nuclear inflammatory cell accumulation, par-ticularly in the molecular layer (Fig. 6) and atthe junction between the molecular and granu-lar layers in most birds. Such focal lesions werealso seen in optic lobes, cerebrums, brainstems,and spinal cords of some birds. Perivascularmononuclear cell infiltration in the white mat-ter continued to be a prominent lesion in thecerebellum at this time also. Such infiltrationswere also seen in optic lobes, cerebral cortices,brainstems, and spinal cords. The cerebellarlesions persisted through day 15, and were pres-ent in all birds with CNS signs. Finally, Weilstrains performed on brain sections taken frombirds with CNS signs failed to demonstratenoticeable demyelinating lesions.

In immunofluorescent studies, the lesions

seen most consistently were again present inthe cerebellum, where they were most prom-inent in Purkinje cells. Here, between 4 and 6days after infection, nearly all birds demon-strated viral antigens in at least a few Purkinjecells, and in some, viral antigens were presentin almost all Purkinje cells in certain folia (Fig.7). By day 7, antigens could be detected inneither Purkinje nor any other cells in theCNS. By ultrastructural methods, viral-specificeffects and viral products were found only inthe cerebella, and then at 5 days postinfection.Sampling errors undoubtedly account for ourinability to locate viral-infected cells else-where. As was expected from the immunoflu-orescent studies, the Purkinje neuron was thecell most often seen to be infected, althoughinfected astrocytes near these neurons were

also observed. In addition, an occasional extra-cellular virion could be seen in the neurophil.Virions were frequently seen to be "budding"from the plasma membrane of Purkinje cellsomas; less frequently this process was ob-

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NEWCASTLE DISEASE 595

FIG. 7. Photomicrograph of the cerebellum from a

chicken processed 5 days after the bird was inocu-lated with NDV. The tissue was subjected to indirectimmunofluorescent procedures specific for Newcastledisease antigens. Several Purkinje neurons and theirdendritic extensions into the molecular layer containviral-specific antigens (x200).

served at dendritic processes. Figure 8 repre-sents a survey micrograph of an infected Pur-kinje neuronal soma. Here, budding Newcastlevirions are present at the plasma membraneand a dense aggregate of nucleocapsid-likestructures is present in the nucleus. The out-lined portion of Fig. 8 is presented in greatermagnification in Fig. 9, where nucleocapsidsare seen within and beneath a budding virion.A similar area in a Purkinje dendrite is shownin Fig. 10. In general, the cytoplasm of infectedneurons was more condensed than that in unin-fected cells and contained swollen organellesand viral nucleocapsids.The dimensions of nucleocapsids within vi-

rions (Fig. 11) were compared with morphologi-cally similar components observed elsewhere inthe cell. Large and small masses of loosely ar-ranged nucleocapsids were seen scattered in thecytoplasm of many neurons (Fig. 12). In addi-tion, nuclei of these cells contained dense accu-

mulation of structures that differed distinctlyfrom condensed chromation or nucleoli (Fig. 13and 14). These forms have the structure anddimensions (16 to 18 nm in diameter) of thenucleocapsids seen in Fig. 11 and 12. The nu-clear forms differed from cytoplasmic nucleo-capsids only in their more orderly and compactarrangement and frequent proximity to the nu-clear membrane. Although such ultrastruc-tural evidence of NDV components in nucleihas not been reported by others, the presence ofantigens observable by immunofluorescentmethods has been claimed (7).

DISCUSSION

This communication establishes the generalvirological and histopathological aspects ofacute velogenic Newcastle disease that precedethe disease of particular interest-the encepha-litis. An initial characterization of this lattersyndrome was also completed. To summarize,after conjunctival and intranasal inoculation,virus rapidly spread to the lungs and induced asevere pneumonitis in all birds, and many suc-cumbed. At this time, significant amounts ofvirus could be isolated from blood, lymphoidorgans and all parts of the brain, and viral-specific products were readily detected inPurkinje neurons. In the brain, the most con-sistently seen and extensive lesions were pres-ent in the cerebellum, where perivascular accu-mulations of round cells in the white matter,focal accumulations of glial cells in the molecu-lar layer, and disappearance of Purkinje neu-rons were prominent. This predilection of NDVfor Purkinje neurons has also been noted byothers (5). A detailed immunofluorescent andultrastructural study of these infected neuronsduring the acute pneumonic phase of the dis-ease indicated that they were productively in-fected. In general, no correlation could be madebetween severity of the generalized illness andextent ofthe viral-induced changes in the CNS.By 9 days after infection, virus could no

longer be detected in any of the pooled tissuesstudied in these birds, and the acute disease inthe survivors had begun to resolve. However, 2to 8 days later, a significant number of thesebirds abruptly developed the severe CNS signsdescribed in the previous section. At the timethat CNS disease became evident, virus couldbe isolated from the brain of only about one-halfof the birds, and then in comparatively smallamounts. In addition, when immunofluores-cent methods were employed at this time, noevidence of virus infection in any cell type inthe brain parenchyma was discernible.

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virions can be seen at the plasma membrane (arrows and outlined area), and a dense aggregate of nucleocap-sid-like structures is present in the nucleus ( x20,000J.

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FIG. 9. The outlined portion ofFig. 8at higher magnification. An accumulation ofnucleocapsids is presentbeneath the budding virion on the right ( 66,000).

FIG. 10. Electron micrograph ofa Purkinje cell dendrite from a chicken infected with NDV. Nucleocapsidsand a budding virion are present (x57,000).

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ing at the plasma membrane can be compared with similar structures present elsewhere in the cell (Fig. 12-14) (x92,000).

FIG. 12. Electron micrograph of Newcastle disease nucleocapsids present in the cytoplasm of a Purkinjeneuron (x92,000).

FIG. 13 AND 14. Electron micrographs ofNDV nucleocapsid-like structures present in the nuclei of Pur-kinje neurons (x92,000).

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NEWCASTLE DISEASE 599

Since the antiviral antibody present in thesebirds undoubtedly neutralized some virus inthe brain both in situ and during preparation ofmaterial for assay, these results are quite com-patible with the hypothesis that a continuingand smoldering virus infection resulted ulti-mately in the appearance of clinical disease.The temporal sequence of events also leads to asuspicion that processes in addition to a directcytolytic effect of virus on various cells in theCNS might be contributing to the clinical dis-ease noted. In this regard, we have been unableto find any pertinent literature relative to para-myxoviruses, but it appears that when mostneurotrophic viruses are given by peripheralroutes, the development of clinically apparentencephalitis correlates temporally with the con-centration of virus in the brain (2, 8, 12, 16).Since the present system yielded different re-sults, it is possible that inflammatory and im-mune responses were playing important detri-mental roles contributing to the development ofNewcastle encephalitis.Taken together, the results indicate that

Newcastle encephalitis is different fronm mumpsmeningoencephalitis and has some similaritiesto measles-virus-induced postinfectious enceph-alitis. However, perivenular demyelination,which is generally considered to be a hallmarkof postinfectious encephalitis (13), was not ob-served to be a prominent feature in the birdsexamined at the time CNS disease developed.Studies concerning additional virological, im-munological, and pathological aspects of thisencephalitis and its more long-term conse-quences are now in progress.

ACKNOWLEDGMENTS

The technical assistance provided by E. D. Scott and V.B. Bastone is gratefully acknowledged.

This investigation was supported by Public Health Ser-vice grant NS-08711 from the National Institute of Neuro-logical and Communicative Disorders and Stroke.

LITERATURE CITED1. Adams, M. H. 1959. Bacteriophages, p. 463-466. Inter-

science Publishers, Inc., New York.2. Albrecht, P. 1968. Pathogenesis of neurotropic arbovi-

rus infections. Curr. Top. Microbiol. Immunol. 43:44-91.

3. Anonymous. 1974. Measles and multiple sclerosis. Lan-cet 1:247-249.

4. Byington, D. P., and K. R. Johnson. 1972. Experimen-tal subacute sclerosing panencephalitis in the ham-ster: correlation of age with chronic inclusion-cellencephalitis. J. Infect. Dis. 126:18-26.

5. Cheville, N. F., H. Stone, J. Riley, and A. E. Ritchie.1972. Pathogenesis of virulent Newcastle disease inchickens. J. Am. Vet. Med. Assoc. 161:169-179.

6. Cook, M. L., and J. G. Stevens. 1970. Replication ofvaricella-zoster virus in cell culture: an ultrastruc-tural study. J. Ultrastruct. Res. 32:334-350.

7. Johnson, C. F., and A. D. Scott. 1964. Cytological stud-ies of Newcastle virus (NDV) in HEp-2 cells. Proc.Soc. Exp. Biol. Med. 115:281-286.

8. Johnson, R.T. 1964. The pathogenesis of herpes virusencephalitis. I. Virus pathways to the nervous systemof suckling mice demonstrated by fluorescent anti-body staining. J. Exp. Med. 119:343-356.

9. Johnson, R. T. 1968. Mumps virus encephalitis in thehamster. Studies of the inflammatory response andnoncytopathic infection of neurons. J. Neuropathol.Exp. Neurol. 27:80-95.

10. Johnstone, J. A., C. A. C. Ross, and M. Dunn. 1972.Meningitis and encephalitis associated with mumpsinfection. A 10 year survey. Arch. Dis. Child. 47:647-651.

11. Miller, H. G., J. B. Stanton, and J. L. Gibbons. 1956.Para-infectious encephalomyelitis and related syn-dromes. Q. J. Med. 25:428-505.

12. Murphy, F. A., S. P. Bauer, A. K. Harrison, and C. W.Washington. 1973. Comparative pathogenesis of ra-bies and rabies-like viruses. Viral infection and tran-sit from inoculation site to the central nervous sys-tem. Lab. Invest. 28:361-376.

13. Scott, T. F. M. 1967. Postinfectious and vaccinial en-cephalitis. Med. Clin. N. Am. 51:701-717.

14. Wear, D. J., and F. Rapp. 1971. Latent measles virusinfection of the hamster central nervous system. J.Immunol. 107:1593-1598.

15. Weiner, L. P., R. T. Johnson, and R. M. Herndon. 1973.Viral infections and demyelinating diseases. N. Engl.J. Med. 288:1103-1109.

16. Zisman, B., E. F. Wheelock, and A. C. Allison. 1971.Role of macrophages and antibody in resistance ofmice against yellow fever virus. J. Immunol. 107:236-243.

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