Cell-fusing activity of visna virus particles

VIILOLOGY 31, 279-288 (19G7)

Cell-Fusing

Activity

DONALD

H. HARTER3

l’he

Rockefeller

of Visna

Accepted

PURn:ELL

ASD

University,

Virus

New November

York,

Sew

Particles”

*

W. CHOPPIN York

10021

3, 1966

Visna virus multiples in a strain of cells (SCP) derived from trypsin-dispersed sheep choroid plexus. After inoculation of 4 TCIDjo/cell, the latent period was E-20 hours and a maximum yield of 160 TCID&cell was obtained at 70 hours. The virus does not multiply in a line of baby hamster kidney (BHKSl-F) cells. Inoculation of BHKSl-F and SCP cell monolayers with 7 TCIDjo and 28 TCIDho/ cell, respectively, induced giant-cell formation within 30-60 minutes, and cell fusion progressed to involve the entire monolayer by 5-6 hours. The large syncytia subsequently disintegrated. The extent of polykaryocyte formation was dependent on virus concentration and was prevented by antiviral antibody. In a potassium tartrate density gradient, virus infectivity and cell-fusing activity both came to equilibrium at a buoyant density of approximately 1.19 g/ml. Virus which had lost its infectivity after ultraviolet irradiation retained the ability to cause cell fusion. Treatment of SCP or BHKBl-F cell monolayers with neuraminidase did not prevent infection or cell fusion. The results indicate that the visna virus particle is capable of inducing alterations in cell membranes leading to cell fusion without virus replication. The possibility of a relationship between the action of the virus on cell membranes and the demyelination which is a feature of the disease it produces is discussed. INTRODUCTION

Visna virus causes a slow, progressive disease of the central nervous system of sheep characterized by perivascular infiltration, glial fibrosis, and demyelination (Sigurdsson et al., 1957, 1962; Pette et al., 1961). It provides a model of a chronic neurological diseaseof proven viral etiology in which the virus is known to be present during the symptomatic phase. Extensive studies

of the disease

in sheep

have

been

1 Supported by Grant AI-05600 from the National Institute of Allergy and Infectious Diseases and by Public Health Service Grant GM-577 from the Institute of General Medical Sciences. 2 An abstract of these studies appeared in J. Clin. Invest. 45, 1020, 1966. 3 Recipient, of Special Fellowship BT-1092 from the National Institute of Neurological Diseases and Blindness. Present address : College of Physicians and Surgeons, Columbia University, New York, New York 10032.

carried out (Sigurdsson et al., 1957; Sigurdsson and P&son, 1958; Gudnaddttir and P&lsson, 1965), and propagation in cells derived from explants of sheep choroid plexus has revealed a number of biological, physical, and chemical properties of the virus (Sigurdsson et al., 1960; Thormar, 1960, 1961, 1963, 1965a, b, c, 1966; Thormar and Cruickshank, 1965). It is a spherical ether-sensit,ive virus, 70-100 mp in diameter, which matures at t’he cell membrane by budding; its nucleic acid has not yet been characterized. In cell cultures derived from explants of the choroid plexus of Icelandic sheep brain, visna virus causes cytopathic changes which include giant cell formation 24-36 hours aft,er infection (Thormar, 1963). This cation

communication of visna virus

describes the repliin cultures derived

from trypsin-dispersed sheep choroid plexus cells, and the rapid, virus-induced fusion 279

HARTER

2so

AND CHOPPIN

of these cells as well as baby hamster kidney cells in which the virus does not multiply. Data will be presented which suggest that visna virus particles possessthe abilit’y to alter cell membranes, leading to cell fusion. The failure of neuraminidase to prevent infection or giant cell formation by visna virus will be reported. MATERIALS

AND METHODS

Cell cultures. Sheep choroid plexus (SCP) cells. Choroid plexi were removed from the lateral ventricles of brains of exsanguinated domestic Hampshire or Suffolk sheep by dissection, minced, and treated with a 0.25 % trypsin solution at 37”. Dispersed cells were decanted into reinforced Eagle’s medium (Bablanian et al., 1965) with 10% fetal bovine serum, and centrifuged at 250 y for 10 minutes at 4”. Cells were resuspended in medium, distributed int’o 250 ml plastic flasks and incubated at 37”. A cell strain prepared in this manner usually survived lo-12 serial passages,and monolayers could be maintained for 3 weeks in reinforced Eagle’s medium with 2% lamb serum. Baby hamster kidney cells (BHK21-F), a heteroploid variant of the BHK21 cell line of Macpherson and St’oker (1962), were obtained from Dr. Sonia Buckley of the Yale Arbovirus Research Unit’, and were propagated as previously described (Holmes and Choppin, 1966). Viruses. Visna virus K485 was obtained from Drs. H. Thormar and P. A. PSLlsson, Institut’e for Experiment’s] Pathology, University of Iceland, and carried through 8 serial passagesin SCP cells. Fifth to 8th passagevirus preparations containing 8.0 X lO”.O to 2.0 X 107.0TCIDSo/ml were used in these experiments. The W3 st’rain of simian virus 5 (SV5) was grown in primary cultures of monkey kidney cells as previously described (Choppin, 1964). The Hickman strain of Newcastle diseasevirus (NDV) was propagated in embryonated chicken eggs. All virus stocks were stored at -60” m&l use. Assay of infective virus. Confluent SCP monolayers in 60-mm plastic petri dishes, 4-6 per dilution, were washed twice wit,h

phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954), pH 7.2, and inoculated with serial tenfold dilut’ions of virus in reinforced Eagle’s medium with 0.5% bovine plasma albumin (Fraction V, Armour Pharmaceutical Co.). After a 3 hour adsorption period at, 37”, maintenance medium was added, and t,he cultures were incubated at 37” in a humidified atmosphere of 5 % COT, and examined after 10, 14, and 21 days for cytopat’hic changes; 50% infect,ivity (TCID,,) end points were calculated by t,he method of Reed and Muench (193s). Plaque assay of SV5 was carried out’ on BHK21-F monolayers using an overlay consisting of reinforced Eagle’s medium, 4% calf serum, 2 % t’ryptose phosphate brot#h, and 0.95 % Difco Noble agar. Cell fusion. Confluent monolayers of SCP or BHK21-F cells in 60-mm plastic petri dishes containing l&mm square coverslips were washed t’wice wit)h PBS, inoculated wit’h virus, and placed at 37” for 2 hours. The inoculum was then removed and maintenance medium added; after 5 or 6 hours coverslips were harvested, fixed in absolut(e methanol, and stained by the May-Griinwald-Giesma method (Jacobson and Webb, 1952). Giant, cell formation was quantitated by count,ing the number of mononuclear cells and polykaryocytes in 10 random microscopic fields on each coverslip at a magnification of 400 tdmes, each field conOainingabout, 50 SCP nuclei or 140BHK21-F nuclei; each cell was given a value of 1 and the number of polykaryocytes was expressed at the percentage of the total number of cells. Other methods of expressing the data were also at’tcmpted, including an index based on the number of nuclei compared to the number of cells, but’ the above met#hodwas found to be a more sensitive measure of the progressof cell fusion. Conceni7-ationof visna virus. Visna virus was concentrated by clarification at 1500 9 for 10 minutes followed by centrifugnt,ion at ‘is,000 g for 5 hours. Pelleted virus was resuspended in one-fifti&h of its original volume in reinforced Eagle’s medium wit,h 0.5 70 BSA and stored at -60” unt’il use. Such preparations contained 1.0 X lO*.O to 1.3 X 10g.OTCIDSO/ml.

CELL-FUSING

.4CTIVITY

Visna virus antiserum. Serum 8643 from a sheep infected with visns virus was kindly furnished by Dr. Thormar. In this laboratory a 1:512 dilution of serum neut#ralizcd 10,000 TCID,, of visna virus. Ultraviolet irradiation. Visna virus was purified by potassium tartrate density gradient centrifugation and suspended in PBS with 0.5 % BSA; 2.0 ml virus was placed in a sterile glass petri dish and exposed to a I
OF

VISNA

281

VllbUS

borate buffer, pH 7.0, prevented agglutination by S hemagglutinating units of the Lee strain of influenza B virus. RESULTS

Growth of Visna Virus in SCP Cells and Failure to Grow in BHK21-F Cells Confluent monolayers of SCP and BHK21-I: cells were infected with visna virus at a multiplicity of 4 TCID,, per SCP ccl1 and 0.6 TCIDso per BHK’Ll-I? cell. (As will be described below, inoculation of cells at higher multiplicit’ies induced rapid cell fusion and degenerat,ion which prevented quantitative studies of virus multiplication.) At intervals, medium and cells were harvested, BSA was added to a concentration of 0.5 %, and the samples were frozen and thawed three times and assayed for infective virus. As shown in Fig. 1, after a latent period

-i \ I3 0 t-

z

E .2 z-7

I 04-

o--a

IOJ-

IO’-

SCP cells BHK21-F

cells

C-+ ‘?

0

I IO

\

\

\

\ I -20

I 30

1 40

f” 50

b 60

I 70

Hours 1. Growth of visna virus baby hamster kidney (BHKZl-F) per cell, and BHKSl-F monolayers FIG.

at 37” in sheep choroid plexus (SCP) cells. SCP monolayers were inoculated at 0.6 TCIDso per cell.

cells, and failure at a multiplicity

to replicate in of 4 TCIDso

282

HARTER

Virus

multiplicity,

AND

CHOPPIN

TCID5&ell

FIG. 2. EfYect

of virus multiplicity on visna virus-induced fusion of SCP and BHKBl-F cells. Monolayers were inoculated wit,h serial twotold dilutions of virus, and polykaryocytes were counted after G hours at 37”.

in SCP cells of 16-20 hours, there was an exponential increase in infective virus from 20 to 36 hours, followed by a more gradual rise. A maximum virus yield of 160 TCIDSo/ cell was obtained at 70 hours. Cytopathic changes marked by t,he appearance of large refractile cells were first noted at 32 hours after infection. These results in cells derived from trypsinized domestic sheep choroid plexus are in general agreement with previous studies of Thormar (1963) on the growth of visna virus in cells derived from explants of the choroid plexus of Icelandic sheep, and in preliminary experiments in this laboratory with such explantderived cells kindly provided by Dr. Thormar, similar virus yields were obtained. Figure 1 also shows that there was no evidence of visna virus multiplication in BHK21-F cells; after 20 hours visna virus could not be recovered from the cell cultures. In other experiments BHKSl-F cells have been maintained for as long as 5 days after inoculation without evidence of virus replicat,ion.

I

I I

I

2

3

I

I

4

5

Hours

FIG. 3. Rates of visna virus-induced fusion of SCP and BHKZl-F cells at 37”. SCP monolayers were inoculated at a multiplicity of 28 TCIDso per cell, and BRKBl-F monolayers at 7 TCID:o per cell.

on coverslips were inoculated with serial two-fold dilutions of virus containing 2.3 X lo5 to 5.6 X 10’ TCIDbO/ml, and after 6 hours at 37” giant cell formation was quantitated. As shown in Fig. 2, the extent of giant cell format’ion was dependent on virus multiplicity. Approximately 4 TCIDS,, per BHK21-F cell caused 50% polykaryocyte formation, whereas about 16 TCIDSO per SCP cell were required; complete cell fusion was obtained at multiplicities of 7 and 28 TCIDQ respectively. The rates of fusion of SCP and BHK21-F cells are illustrated in Fig. 3. Confluent monolayers were inoculated with 5.7 X 10’ TCID,, of visna virus, representing a multiplicity of 7 TCID,, per BHK21-F cell and 60 TCID,, per SCP cell, and polykaryocyte formation was determined on coverslips harvested at 30-minute intervals. Significant cell fusion was detected Virus-Incluced Cell Fusion 30-GO minutes after inoculation and was Early experiments indicated that. inocu- complete in both cell t’ypes by 5 hours. lation of SCP or BHKBl-F cells with visna Initially, polykaryocyte formation was virus at moderate multiplicities caused more rapid in BHK21-F monolayers. Thus rapid cell fusion. To determine the effect of BHK21-F cells appear to be somewhat virus concentration on cell fusion, repli- more sensitive to virus-induced cell fusion cate monolayers of SW and BHK21 cells in terms of both the virus/cell multiplicity

CELL-FUSING

ACTIVITY

OF VISNA

VIRUS

4 and 5. Fusion of SCP and BHKBl-F cells by visna virus at. 37”. Photomicrographs take !n 6 inocrdation. May-Griinwald Giemsa stain. Magnification: X 150. Fig. 4a. Control ECP Fig. 410. SCP monolayer inoculated with 60 TCIDja per cell. Fig. 5a. Control BHK2 1-F monolayer inoculated with 7 TCID:o per cell. III lonolayer. Fig. 5b. BHKSl-F FIGS.

111JWS after m onolayer.

required and the rapidity of fusion by a given concentration of virus. The extreme sensitivity of t’hesecells t’o fusion by simian virus 5 (SV5) has been previously described (Holmes and Choppin, 1966). W&h both SCP and BHK21-F cells the curve depicting cell fusion is biphasic; tbe second, slower phase probably reflectIs the fact’ bhat ext,ensive fusion had already occurred, great,ly reducing the number of cells so that further fusion necessarily involves progressively fewer and larger giant cells. Although, the rates of fusion appear similar lat,e in these experiments, the significance of this similarit,y is doubtful since, in terms of amount of membrane involved, most of the cell fusion had occurred earlier. As fusion progresses, SCP cells form large stellate polykaryocytes w&h many nuclei frequently arranged in a circular pattern in the central part’ of t)he cell. In BHK21-F

syncyt’ia, the nuclei are often arranged in long parallel rows similar to those found in these cells after inoculation with SV5 (Holmes and Choppin, 1966). In bot’h cell types, visna virus induces fusion which progresses to involve essentially all cells in a giant syncyt’ium as shown in Figs. 4 and 5. These syncytia usually degenerate wit’hin 6-S hours. These results indicate that visna virus in sufficient’ concentration can rapidly induce massive fusion of cells before new infective virus is produced, and furthermore that it can induce fusion of cells in which it. does not mult.iply. Association visna

of Cell-Fusing Vi;rus Particle

Activity

with

the

To purify the virus, determine its density, and demonst’rate t#hat t’he ability to induce (sellfusion is a propert,y of the visna virion,

284

HAllTElL

AND

CHOPPIPU’

I.28

<151--/,-

20 t

.ll...-\.I4

2

8

I2

16

20

24

Fraction FIG. 6. Equilibrium zonal centrifugation of infective visna virus in potassium tartrate. Virus partially purified by differential suspension, centrifugation, was placed on a preformed gradient and centrifuged at 100,000 g for 3 hours at 4”.

virus was subjected to equilibrium zonal centrifugation in potassium tartrat’e, and the infectivity and the cell-fusing activity of the fractions obtained were determined. Virus concentrated and partially purified by differential centrifugation was layered onto a preformed gradient of 5-50 % w/w potassium tartrate in 0.1 M phosphate buffer, pH 7.0, containing 0.001 M EDTA and centrifuged in an SW39 swinging bucket rotor at 100,000 g for 3 hours at 4”. Threedrop fractions were collected from below, and the infectivity titers were determined. The results of such an experiment are shown in Fig. 6. Over 90% of the infective virus placed on the gradient was recovered in a discrete bluish-white band located 36 mm from the bottom of the tube at a density of approximat’ely 1.19 g/ml. A second band of flaky opalescent material which did not contain significant infectivity was noted 23 mm from the bottom of the tube. The nature of this band has not yet been determined. To determine cell-fusing activity, sixdrop fractions were used to obtain enough material for assay. To remove potassium

4

6 Fraction

8

IO

12

FIG. 7. Cell-frlsing activity of visna virus subjected to equilibrium zonal centrifugation in potassium tartrate. Fractions were inoculated onto BHKPI-F monolayers and polykaryocytes counted after 6 hours at 37”.

tartrate, fractions were centrifuged at 100,000 g for 3 hours, and the sediment was resuspended in reinforced Eagle’s medium with 2% lamb serum. The resuspended fractions were then added to replicate monolayers of SCP and BHK21-F monolayers. Polykaryocyte formation was quantitated after 6 hours. Figure 7 shows result’s obtained on BHK21-F cells; similar result’s were obtained with SCP cells. The capacity to induce giant cells was maximal in the gradient fraction which contained the peak concent’ration of infect.ive virus. Additional evidence that cell-fusion is associated wit’11 t#he visna virion is shown in Table 1. Visna virus antiserum obtained from sheep infected in Iceland prevented the fusion of both BHKSl-F cells and SCP cells by visna virus which had been propagated in this laboratory and purified by gradient centrifugation. Cell Fusion by UV-Iwadiated

Virus

Purified visna virus was exposed to ultraviolet light and infectivity and cell-fusing activity were determined. As shown in Table 2, virus irradiated for 5 minutes lost > 99% infectivity but retained cellfusing activity, indicating that infectivity

CELL-FUSING TABLE

ACTIVITY

Treatment

of virusa

Normal sheep serum Visna virus antiserum

per cells”

G7 2

2

EFFECT OF ULTRAVIOLET IRRADL~TION ON THE CELLFUSING ACTIVITY OF Vls~a VIKGS UV (Eip$Tsta

Surviving:infective virus

Polykaryocytes (‘,T; of total cells)b (72)

BHKZl-I? cells

SCP cells

100 0.36 0.0005

0

5 15

a 2.8 X 107 TCIDso 15-watt 15 cm.

Westinghouse

b Counted

100 100 18 virus Sterilamp

Fith

at a distance

9.3 x 105 8.7 x 105

NDV

Buffer Neuraminidase

1.5 x 109 1.1 X 108

n Cells were t,reated for 1 hour at 37” with calcium borate buffer, pH 7.0, or 25 units of neuraminidase, washed 3 Gmes with PBS, pII 7.2, and inoculated with virus. TABLE 4 FAILURE OF NEURAMINIDASE TREATMENT OF CELLS TO PREVENT VISNA VIRUS-INDUCED POLYKAKYOCYTE FORMATION Polykaryo-

Cells

Visna

a of

for fusion of cells by visna Irradiation for 15 minutes caused a marked reduction in cell-fusing activity as well as infectivity.

is not required virus part#icles.

Lack of E$ect of Newaminidase Treatment of Cells on Infection and Cell Fusion Induced by Visna Virus surface

Buffer Neuraminidase

Treatment of cells” c@es (% of total cells)b

SCP

Visna

BHK21 -F

sv5

BHK21-F

6 hours after inoculation.

Because of t’he apparent similarity structure

of visna

virus

to t’hat

Infective virus (TCIDdnl)

of cellsa

Visna

Virus

100 100 12

irradiated

285

Treatment

100 8

a Mixtures of 5.0 X lo7 TCIDjO of virus and 1:64 diluted serum were incubated for 1 hour at 37” before addition to cells. b Counted G hours after inoculation. TABLE

VIRUS

Virus

BHKZl-I; cells

SCPicells

VISNA

TABLE 3 FAILURE OF NEURAMINIDASE TREATMEXT OF CELLS TO PREVENT INFECTION OF SEIEEP CHOROID PLEXUS CELLS BY VISNA Vmus

1

PREVENTION OF CELLFUSION BY ANTIBODIES AGBINST VISNA VIRUS Polykaryocytes, cent of total

OF

a Cells

were

treated

Buffer

100

Neuraminidase Buffer Neuraminidase Buffer Neuraminidase

100 100 100 100 G

for

1 hour

at 37” with

cal-

cium borate buffer, pII 7.0, or 25 units of neuraminidase, washed 3 times with PBS, pII inoculated with 6.5 X 1OS.O TCIDSO of h Counted 5 hours after inoculation.

tative

studies

of visna

difficult,;

however,

neuraminic

acid

virus

adsorption

7.2, and virlls.

are

the possible role of

residues

in

visna

virus

in

adsorpt’ion could be approached by de-

of

t,ermining the effect, of neuraminidase treat’ment of cells on (1) infection of SCP cells

myxoviruses (Thormar and Cruickshank, 1965), t,he possibilit’y of a neuraminic acid-cont,aining receptor for the virus was investigated. Hemagglutination by visna viruses has not so far been found; in addition to the many species of erythrocytcs em-

and (2) fusion of SCP and BHKSl-F cells. As controls to confirm the destruction by neuraminidase of myxovirus receptors on these cells, the effects of t’he enzyme on the infection

of SCP cells by Newcastle

disease

ployed by Thormar (1965b), we have virus, and on the fusion of BHE<21-E’ cells examined rhesus monkey, pigeon, duck, by SVS were also tested. goose, ox, swine, goat, and turkey cells As shown in Table 3, neuraminidase treatwithout success. Due to the lack of a model syst,em such as red cell agglutination, quanti-

ment of SCP cells failed to prevent infection by visna virus, but did reduce t’he in-

286

HARTEH

AND

CHOPPIN

semially an extension of the cell membrane of the oligodendrocyt8e, a glial cell which presumably forms and maintains the functional integrity of myelin (Peters, 1960; Bunge et al., 1962). There are at least two possible pathways by which visna virus might lead to demyelination: (1) the virus could multiply in t’he oligodendrocytes causing cell injury or death resulting in demyelination, (2) alternatively virus or viral components produced in neighboring cells or elsewhere might alter myclin or the membranes of the oligodendrocytes by a direct effect; this could result in either dest’ruction or alterations of such a nature as to render the membranes immunogenic. DISCUSSION That multiplication in the oligodendrocyte The present studies have provided evimight not, be necessary for injury of memdence that visna virus particles possess the branes is suggested by the effect, of visna ability to alter cell membranes, leading to virus on BHK21-F cells in which it does cell fusion and event’ual disintegration. not replicate. It should be emphasized that The association of cell-fusing activity with visna virus is present in the central nervous the virus particle is indicabed by the sedisystem of sheep during the symptomatic mentation of this activity with infective phase of t’he disease when demyelination virus in a density gradient,, by t’he inhibition occurs; it has been isolated from choroid of cell fusion by antiviral antibodies, and plexus, spinal fluid blood, and other Oissues by the dependence of cell fusion on virus up to four years after infection (Gudnadotconcentration. That infective virus or viral tir and Palsson, 1965). Thus the oppormultiplication is not required for cell fusion tunity exists for direct, action of t,he virus is shown by fusion of BHK21-F cells in on myelin or cell membranes. which the virus does not multiply, by fuIt is also of interest, that two other animal sion of BHKBl-F cells and SCP cells wit,h viruses which can cause encephalomyelitis UV-irradiated virus, and by fusion of SCP accompanied by demyelination and which cells as early as 30 minutes after inocula- are present in the neural tissue at the time tion, many hours before new virions are the lesions develop, i.e., canine distemper produced. Thus, the visnu virus particle (Hurst et al., 1943; Rockborn, 1953), and joins a number of other virions which are mouse hepatitis virus (Cheever et al., 1949), capable of inducing cell fusion. This group also possess cell-fusing activity. Furthcrof viruses includes both RNA and DNA more, giant cell formation is prominent in viruses, e.g., the parainffuenaa-mumps- cells infected with measles (Enders and Newcastle disease subgroup of myxoPeebles, 1954; Roberts and Bain, 1958; viruses, measles, and herpes (Roizman, 1962; Warren et al., 1962; Kahn, 1965). Cascardo and Karzon, 1965) and mumps Although the precise mechanism of cell (Henle et al., 1954) viruses, agenm which fusion by visna virus or other viruses is can induce “post’infection” encephalomyeunclear, it is of interest to consider whether litis in man. Ot#her virus infections which the abilit’y of the virus to induce the altera- may be associated with this complication, tions in cell membranes could be relat,ed although more rarely, varicella, vaccinia, to the demyelination which is prominent and smallpox, produce cell fusion in vivo in visna-infected neural bissue. Myelin is a or in cell culture. There thus appears to membranous structure which, in the central be sufficient’ reason to explore further the nervous system, has been shown to be es- possible role of the action of viruses on cell fectivity titer of Newcastle disease virus in these cells by more than tenfold. Table 4 indicates that neuraminidase treatment of SCP and BHK21-F cells did not prevent the fusion of these cells by visna virus, but did prevent SV5-induced cell fusion. Thus, no evidence was obt,ained t’hat visna virus adsorbs to neuraminic acid-containing receptors similar to t’hose of myxoviruscs; however, the possibility cannot be completely excluded that such receptors exist, but that the neuraminic acid residues were either insusceptible or inaccessible to the neuraminidase employed in t,hese experiment,s.

CELL-FUSING

ACTIVITY

membranes in the pathogenesis of chronic demyelinating diseases. There are of course ways that visna infection might lead to demyelination other than direct action on membranes. Gudnaddttir and PAlsson (1965) have suggested that a reaction occurring on the surface of infected glial cells between neutralizing antibodies and viral antigen results in secondary demyelination. It is perhaps significant that viruses which may be associated with “postinfection” encephalomyelitis in man, e.g., measles, mumps, varicella, smallpox, and vaccinia, though a diverse group morphologically and chemically, possess a common feature, the presence of a lipid containing envelope which is probably derived at least in part from the cell membrane. Possible mechanisms whereby such viruses might play a role in the pathogenesis of encephalomyelitis either by direct injury or in a pathogenic immune react’ion have been discussed elsewhere (Choppin, 1966). ACKNOWLEDGMENT The authors wish to Funk and Mr. Salvatore technical assistance.

thank Mrs. Karen K. Spatola for excellent

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HARTER

ROCKBORN, G. (1958). tissue 492.

ROIZMAN,

culture.

Arch.

Canine distemper ges. Vimsforsch.

AND

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