INFECTION AND IMMUNITY, Sept. 1975, p. 614-620Copyright © 1975 American Society for Microbiology

Vol. 12, No. 3Printed in U.S.A.

Growth of Measles Virus in Cultures of Rat Glioma CellsKIYOTO NAKAMURA,* MORIO HOMMA' AND NAKAO ISHIDA

Department of Bacteriology, Tohoku University School of Medicine, Sendai, Japan

Received for publication 27 February 1975

The interaction of measles virus with RG-6 cells derived from rat glioma wasinvestigated. When a culture of RG-6 cells was infected with measles virus, thesynthesis of viral antigens was detected in very few cells, at most 5%7. Theapparent resistance to measles virus infection was also repeatedly found in all ofthe subclonal cells derived from RG-6 cells. Although all of the virus-synthesizingcells had the ability to form plaques on Vero cells, they produced only a reducedamount of infectious virus, i.e., 0.1 plaque-forming units per cell. These resultsimply the existence of some mechanism that regulates growth of measles virus incultures of RG-6 cells. The transmission of genetic material of measles virus frominfected RG-6 cells to Vero cells was not inhibited in the presence of antiviralserum. This fact may provide a basis for interpretation of the persistence of virus,in the presence of antibody, in patients with subacute sclerosing panencephalitis.

Little is known about the pathogenesis ofsubacute sclerosing panencephalitis (SSPE),though involvement of measles virus has beenshown by several authors (5-7, 15). With regardto the pathogenesis of SSPE, the following twofacts should be considered. (i) The isolation ofthe virus was only successful when trypsin-dis-persed brain cells of the patients were cocul-tivated with susceptible cells (1, 8, 9, 12). Thisseems to show that infectious virus is notproduced in the brain cells of the patients. (ii)High titer levels of antibodies against measlesvirus were present both in sera and cerebrospi-nal fluids of patients with SSPE (5). It is ofinterest, therefore, to know how SSPE viruspersists in the brain cells of the patients in thepresence of antibody.This report is concerned with the interaction

of measles virus with rat glioma cells. We willshow that at any time during infection only apart of the cells were infected and viral produc-tion in the infected cells was largely restricted.We also describe the effect of antiviral serum onthe transmission of measles virus from infectedRG-6 cells to uninfected Vero cells.

MATERIALS AND METHODSTissue culture: ;s. The RG-6 cell line estab-

lished from rat glioi.a by Benda et al. (3) was kindlysupplied through T. Nakazawa of Keio UniversitySchool of Medicine, Japan. The cells in monolayerwere grown in Eagle minimum essential medium(MEM) with 0.29%, L-glutamine supplemented with10% unheated bovine serum. For virus infection,MEM with 2% heated horse serum (MS) was used.

'Present address: Department of Bacteriology. YamagataUniversity School of Medicine, Yamagata. Japan.

HeLa-S3 cells were grown in a medium consisting of0.5% lactalbumin hydrolysate, 0.1%Y. yeast extract inEarle balanced salt solution, and 10%7c bovine serum.Through the course of infection, the growth mediumwas replaced with yeast extract in Earle balanced saltsolution with 2% heated horse serum. Vero cells weregrown in MEM with 10% calf serum, which wasreplaced by MS when infected.

Virus. The Edmonston strain of measles virus,which was passaged four to ten times into HeLa-S3cells, was used. The virus was recovered from theinfected cells after three cycles of freezing and thaw-ing followed by centrifugation at 900 x g for 15 min.The clear supernatant fluids were stored at -80 Cuntil used. The infectivity titers ranged from 1062to 107 2 mean tissue culture doses (TCD55)/ml.

Infectivity titration. The infectivity was measuredeither by plaque formation or by tube titration. Forplaque formation, a monolayer culture of Vero cells ina petri dish (60 mm in diameter) received 0.2 ml ofsamples to be tested. After incubation for 2 h at roomtemperature, the inoculum was removed and theculture was overlayed with 5 ml of agar mediumconsisting of MEM with 2% heated calf serum. Thesecond overlay was made 4 days after infection, andthe plaques were counted on day 8 by staining withneutral red in Hanks solution (1:10,000). For tubetitration, the tube cultures of HeLa-S3 cells received aseries of 10-fold dilutions of the sample, and theTCD,O was determined by the appearance of cyto-pathic change on day 14 according to the method ofReed and Muench (14). Three tubes were used perdilution.

Immunofluorescence test. Immunofluorescent cellcounting was done according to the method of Ka-shiwazaki et al. (10). Cover-slip (6 by 30 mm) culturesof RG-6 cells prepared in petri dishes received 0.05 mlof test samples 24 h after the cells were seeded at aconcentration of 1.25 > 106 cells per dish. Afteradsorption for 2 h at 37 C in a CO2 incubator, thecover slips were washed and immersed in new petri

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dishes containing 5 ml of MS, and incubation wascontinued. At appropriate intervals, the cover slipswere removed from the petri dishes, washed in phos-phate-buffered saline, pH 7.2, dried, fixed with car-bon tetrachloride, and stained with fluorescent anti-body by the indirect method. An adult human serumwith staining titer of 1:80 was used as the first serum.Goat anti-human immunoglobulin G labeled withfluorescein isothiocyanate (Hyland Co.) was diluted80-fold in phosphate-buffered saline and used as thesecond serum.

Uninfected RG-6 cells were never stained by thisprocedure, and treatment of measles-infected RG-6cells with rabbit antiviral serum before addition of thefirst serum completely inhibited staining of the in-fected cells, which confirmed that this procedurespecially stained measles antigen in infected cells.However, the antigen detected by this stainingmethod was not characterized. The fluorescent cellswere counted at a magnification of x400. Fifty fieldswere usually counted per cover slip, and the meannumber of fluorescent cells per field was obtained.The percentage of cells with fluorescence was deter-mined from counts of 1,000 cells.

Preparation of cell-associated virus. An infectedmonolayer culture was washed thoroughly and thenreceived the original volume of MS. After three cyclesof freezing and thawing, the resulting cell debris wasremoved by light centrifugation and the clear super-natant fluids were assayed for infectivity.

Antiviral serum. A rabbit was inoculated intrave-nously with 6 ml of HeLa-S3 cell-grown measles virusat 106 8 TCD,O/ml, which was followed by five succes-sive weekly subcutaneous injections of the virus withthe same amount of Freund incomplete adjuvant. Theanimal was bled 1 week after the last injection and theserum was inactivated by heating at 56 C for 30 min.To estimate the neutralization titer of the antiviralserum, serial twofold dilutions of the serum in 0.2 mlwere mixed with equal volumes of 100 TCDSO ofmeasles virus, and after incubation at room tempera-ture for 30 min, 0.2-ml aliquots of the mixture wereadded to the tube culture of HeLa cells. The titer,determined after incubation of the culture at 37 C for14 days, was 1:512. The serum was diluted 40-fold inMS before use.

Cloning of RG-6 cells. RG-6 cells dispersed by0.1% trypsin plus 0.1% ethylenediaminetetraacetate(EDTA) were plated into the petri dishes at aconcentration of 20 cells per dish and cultivated in aCO2 incubator for 2 to 3 weeks. A total of 40 colonieswas inserted into test tubes and grown separately.

RESULTSImmunofluorescent study. When a mono-

layer culture of RG-6 cells was infected withmeasles virus at a multiplicity of infection(MOI) of 10 TCD5O per cell, visible cytopathicchange did not appear even after a prolongedincubation period of 2 weeks. Immunofluores-cent staining, however, detected the synthesisof viral antigens in a limited number of cells, 5%at most. Fine granular fluorescent spots ap-peared in the cytoplasm 24 h after infection; the

intensity of the fluorescence was increasedthereafter, and the whole cytoplasm becamefilled with homogeneous fluorescence by 48 hafter infection. On the contrary, intranuclearfluorescence could not be detected throughoutthe course of infection. At any time, the distri-bution of the fluorescent cells was fairly evenand focus-like appearance of the fluorescentcells was rarely found. The time course of theappearance of the fluorescent cells is illustratedin Fig. 1. The fluorescent cells began to appearat 24 h, followed by a rapid increase in thenumber of fluorescent cells. Maximum levelswere reached 30 to 48 h after infection, followedby a gradual decrease. The decrease was due toselective detachment of the fluorescent cellsfrom cover slips caused by degeneration.Growth of measles virus in RG-6 cells. The

tube cultures of RG-6 cells were inoculated with0.2 ml of virus suspension at an MOI of either 6or 0.06 TCD50 per cell and incubated at 37 C for2 h. After washing the unadsorbed virus, 1 ml ofMS was added to each tube and incubation wascontinued at 37 C. At the times indicated, bothfree and cell-associated viruses were tested forinfectivity on the monolayer cultures ofHeLa-S3 cells.The virus yields were very poor with either

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Time after infection (h)FIG. 1. Time course of the appearance of fluores-

cent cells. The cover-slip cultures of RG-6 cells wereinfected with measles virus at a multiplicity of 10TCD,O per cell. The immunofluorescent staining wasmade at the indicated times, and the number offluorescent cells was determined by the methoddescribed previously (10).

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616 NAKAMURA, HOMMA, AND ISHIDA

MOI and were fairly multiplicity dependent(Fig. 2). Even in the cultures infected at anMOI of 6, the maximum titer of the cell-asso-ciated virus was only 102-8 TCD50/ml and thevirus yield per fluorescent cell was roughly cal-culated to be less than 1.Formation of infectious centers by the fluo-

rescent cells. To further examine the poor yieldof infectious virus in RG-6 cells, the followingexperiment was conducted to see whether all ofthe fluorescent cells would become infectiouscenters. The infection of RG-6 cells in tubeculture with measles virus at an MOI of 4TCD5O per cell was made as described above. Atappropriate intervals, the infected cultures weretaken out and divided into two groups. With thefirst group, the cells were dispersed by 0.1%trypsin and 0.1% EDTA and plated on themonolayer cultures of Vero cells in petri dishesto determine their ability to form plaques. Withthe remaining group, both the free and thecell-associated viruses were prepared and theinfectivities were measured on the monolayerculture of Vero cells by the plaque method. Inparallel with these experiments, the replicating

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24 48 72 96

Time after infection (h)

FIG. 2. Growth of measles virus in RG-6 cells. Thetube cultures of RG-6 cells were infected with measlesvirus at a multiplicity of either 6 or 0.06 TCD,0 per

cell. At various times after infection, fluid virus andcell-associated virus were prepared as described in thetext, and the infectivities were titrated on the repli-cating tube cultures of HeLa-S3 cells by the dilutionmethod. Symbols: (0), Cell-associated virus at an

MOI of 6; (A), fluid virus at an MOI of 6; (0),cell-associated virus at an MOI of 0.06: (A), fluidvirus at an MOI of 0.06.

tube cultures were taken at 48 h after infectionto determine the exact number of infected cellsper tube. The cells were dispersed by trypsinand EDTA and washed by light centrifugation.The final cell suspension was smeared on thecover slips, and the number of the infected cellsper tube was calculated on the basis of the ratioof immunofluorescent cells to total cells (Fig. 3).The number of the infectious centers was fairlyconstant until 48 h after infection. The max-imum number of infectious centers was 20,050per tube, which was recorded at 32 h afterinfection, and compared with the number ofimmunofluorescent cells per tube, 21,560, sug-gesting that all of the fluorescent cells would actas infectious centers. In addition, from theseresults the amount of virus yield per infectedcell was calculated, and the value was again aslow as 0.1 plaque-forming units (PFU).

Susceptibility of subclonal RG-6 cells tomeasles virus infection. The preceding resultsmight suggest that there is a genetic difference

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24 48 72

Time after infection (h)FIG. 3. Formation of infectious centers by virus-

synthesizing RG-6 cells. The tube cultures of RG-6cells were infected with measles virus at an MOI of 4TCD,O per cell, and the infected cultures were dividedinto two major groups. At the indicated times, thecells in the first group were dispersed by 0.1 % trvpsinplus 0.1% EDTA. After washing by centrifugation, theresulting cell suspension was plated on the monolayerculture of Vero cells to form plaques by the methoddescribed in the text. The number of plaques formedby the cells in the whole tube is plotted (0). With thesecond group, both fluid virus (A) and cell-associatedvirus (A) were assayed for infectivitity as described inthe text.

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MEASLES VIRUS INFECTION IN RAT GLIOMA CELLS 617

between fluorescent and nonfluorescent cells.Subclones derived from RG-6 cells were there-fore examined for susceptibility to measles virusinfection. A total of 40 subclones was grown intubes, and each was infected with 1060 TCD5O ofmeasles virus. At 48 h after infection, the cellsin each tube were dispersed and smeared oncover slips, and the immunofluorescent cellcounting was done. Fluorescent cells were re-covered from all of the tubes, but the ratio offluorescent cells to total cells in each culturewas again very low, ranging from 1 to 4%. Theresults clearly show that the culture of RG-6cells was not a mixture of cells different insusceptibility to measles virus infection.Adsorption of measles virus onto RG-6

cells. An experiment was conducted to seewhether the rate of adsorption of measles virusto the culture of RG-6 cells in which the virusgrowth was largely restricted was different fromthat found in the culture of HeLa-S3 cells inwhich virus growth occurred normally. Bothcultures of RG-6 cells and HeLa-S3 cells inmonolayer were prepared in tubes and inocu-lated with 1050° TCD,O of the virus in 0.2 ml ofMS, and the adsorption was made at 37 C. Atthe times indicated, the culture fluids con-taining the unadsorbed virus were removed andthe infectivity was measured by the plaquemethod. The rate of adsorption of measles virusonto the culture of RG-6 cells was quite similarto that observed with HeLa-S3 cells, indicatingthat the adsorption,may proceed normally withevery RG-6 cell (Fig. 4).

Effect of cell density in culture on theefficiency of virus infection. An analysis wasmade to explore the poor efficiency of measlesvirus infection in the culture of RG-6 cells. RG-6cells were seeded on different days into petridishes containing the cover slips. The cover-slipcultures at days 1 (sparse culture) and 5 (denseculture) were infected simultaneously with 105 2TCDSO of measles virus. At the times indicated,the cover slips were withdrawn from the sparseand dense cultures, respectively, the immuno-fluorescent staining was done, and the numberof the fluorescent cells per field was counted.The maximum number of fluorescent cells inthe sparse culture was three times greater thanin the dense culture (Fig. 5). Such a phe-nomenon, however, was not observed wheneither HeLa-S3 or Vero cells were infectedwith measles virus.Transmission of virus in the presence of

antibody. Although virus yields of RG-6 cellswere shown to be as low as 0.1 PFU perfluorescent cell, all of the fluorescent cellsseemed to become infectious centers whenplated on Vero cell monolayers. Then it was ofinterest to know the mode of transmission of the

virus progeny from the infected RG-6 cells tothe uninfected Vero cells. The tube culture ofRG-6 cells was infected with measles virus at anMOI of 4 TCD,O per cell. At 7 h after infection,the antiserum was added to the culture andincubation was continued for another 30 min at37 C. The infected cells were dispersed bytrypsin, taken up into the medium containingantiviral serum, plated on the cover-slip cul-tures of Vero cells, and allowed to settle for 2 hin a CO2 incubator. The cover slips were thentransferred into new petri dishes, and furtherincubation was made in the presence of anti-body. On day 3, the cover slips were stainedwith fluorescent antibody. Syncytia withfluorescence developed under this condition(Fig. 6), and the number of syncytia was fairlyproportional to that of the infected RG-6 cellsinoculated. The infectivity of the culture fluids,however, could not be detected, whereas aninfectivity titer of 104-8 TCD50/ml could berecovered from the control experiment in whichantibody was omitted. These results revealedthat the transmission of the genetic material ofthe virus from infected RG-6 cells to Vero cellsactually occurred even in the presence of anti-body that was known to neutralize 1060° TCDSOof measles virus.

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Incubation time (h)

FIG. 4. Adsorption of measles virus onto RG-6 cells.Monolayer cultures of RG-6 cells (-) and HeLa-S3cells (0) grown in tubes were infected with 1050TCDSO of measles virus in a 0.2-ml amount andincubated at 37 C. At the times indicated, unadsorbedvirus in the fluids was removed and the infectivitieswere assayed by the plaque method as described inthe text.

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618 NAKAMURA, HOMMA, AND ISHIDA

DISCUSSION9 We have demonstrated that when a culture

of RG-6 cells was infected with measles virus,8 | the synthesis of viral antigens occurred in a very

limited portion of the cells, 5% at most, and the7 | \ production of infectious virus per virus-synthe-

sizing cell was as low as 0.1 PFU. Although the" 6 \ majority of the remaining RG-6 cells were

6 protected from the infection by some unknownmechanisms, these uninfected cells were shown

,,5 - \ not to be genetically different from infectedcells in susceptibility to measles virus. In fact,

o4 / \ 40 subclones randomly selected from the origi-nal RG-6 cells were found to be equally suscep-

O 3 - tible to infection with measles virus. Further-more, adsorption of the virus to RG-6 cells

ffi 2 ); / \ proceeded at a rate similar to that obtainedz with sensitive HeLa cells.

Previous reports about the interaction ofmeasles virus with cell originated from nervous

1 2 24 36 48 60 72 number of cells on the day of use amounted to 1.25 x106 and 7.5 x 106 cells per dish for the sparse (0) and

Time after infection (h) dense (0) cultures, respectively. The cover-slip cul-FIG. 5. Effect of cell density on the appearance of tures from both groups were infected simultaneously

fluorescent cells. RG-6 cells were plated into petri with 105.2 TCD5o of measles virus. Immunofluorescentdishes containing cover slips. One-half of the cultures staining was made at the times indicated, and thewas prepared 5 days ahead of the remaining cultures, number of fluorescent cells was determined as de-which were prepared on the day of infection. The scribed (11)..-_ ~~. ->s.T......................4 _r.^^.

FIG. 6. Syncytium formation by inoculation of infected RG-6 cells on Vero cells in the presence of antibody.The cultures of RG-6 were infected with measles virus at a multiplicity of 4 TCD5O per cell. At 7 h after infec-tion, antiviral serum was added to the cultures and incubation was made for 30 min. The infected cells werethen dispersed by trypsin and suspended in 1 ml of antiviral serum-containing medium. A 0.05-ml amount ofthe cell suspension was inoculated on the cover-slip culture of Vero cells for 2 h. The cover slips were thenplaced into new petri dishes and incubation was continued in the presence of antiviral serum. On day 3 afterinoculation of the infected cells, immunofluorescent staining was made. Magnification, x200.

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MEASLES VIRUS INFECTION IN RAT GLIOMA CELLS 619

tissues are apparently in conflict with ourresults in some respects. A culture of hamsterdorsal-root ganglion has been shown to supportthe growth of the Edmonston strain of measlesvirus for long periods with a limited amount ofvirus production (13). Similar results have beenobtained with human fetal brain cells wheninfected with either the Shwarz or the Becken-ham 31 strain (16). Recently, establishment oflong-term infection of African green monkeybrain cells was successfully made with theShwartz strain but not with the Edmonstonstrain (2). All of these observations may revealvariable interactions between different braincells and different measles virus strains. Onecommon feature between these observations is,however, that the growth is considerably re-stricted in the brain cells, suggesting that nerv-ous tissue cells have similar mechanisms forregulating the growth of measles virus. A reportdemonstrating a nonspecific inhibitor againstmeasles virus growth in brains of rats andhuman beings appears to support the abovepostulate (4).A regulatory mechanism, if exist in the nerv-

ous tissue, may not be a single entity. Thepresent experiments may have revealed at leasttwo different mechanisms in the culture of RG-6cells. The first one may function at an earlierstage of multiplication since the majority of thecells were neither participating in the synthesisof viral antigens nor able to become infectiouscenters, although measles virus was adsorbedonto them. This type of regulation seems to beinfluenced by the physiological condition ofRG-6 cells because the appearance of antigen-synthesizing cells was more restricted in thedense culture than in the sparse culture. Thesecond one may function at a later stage ofinfection. This is based on the fact that everyantigen-synthesizing cell produced infectiousvirus only in greatly reduced amounts. Thistype of regulation at a late step has beenreported in infection of hamster embryo fibro-blasts with measles virus (11). Two types ofinfection, lytic and latent, were simultaneouslyfound by these authors (11) when primary ham-ster embryo fibroblasts were infected with theSchwarz strain. This may also reveal the di-verse reactivity of the cells to measles virus. Intheir established latent culture, 30 to 50% ofthe cells were found to be synthesizing virus an-tigen. In our case, an attempt to establish along-term latent infection was unsuccessful.Subcultures of the infected RG-6 cells resultedin the disappearance of antigen-synthesizingcells shortly after the passage. Thus the regu-latory mechanism of RG-6 cells seems to befairly potent, either single or multiple. A final

conclusion on such a mechanism, however, isleft for further studies because neither theSchwartz nor the rat-adapted Edmonstonstrain was examined in RG-6 cells.The transmission of the genetic material of

measles virus from infected RG-6 cells to unin-fected Vero cells was not prevented by antiviralserum. Since no virus infectivity could be re-covered from the fluid of the culture in whichantiviral serum was contained, it seemed cer-tain that the transmission of viral genome in thepresence of antibody was not mediated throughthe virus released from infected RG-6 cells.Further, the potency of the antiviral serum washigh enough to inactivate as much as 1060TCD,O of measles virus at the concentrationused, whereas the infectivity associated withinfected RG-6 cells was calculated to be as lowas 68 PFU per cover-slip culture; these factsstrongly suggest that involvement ofthe cell-associated virus in the transmission wasalso unlikely.As the mode of transmission of genetic mate-

rial in which neither released nor cell-associatedvirus is involved, three mechanisms might beconsidered: transmission via intercellular proc-esses, direct transmission by cell fusion, andingestion of infected RG-6 cells by Vero cells. Bywhich of them the virus transmission from RG-6cells to Vero cells was mediated remains to bedetermined. In any case, however, the failure ofantibody to block the transmission of measlesvirus genome from cell to cell may reflect thefact that measles virus persists for a long time inthe brains of SSPE patients in spite of thepresence of high antibody titer in serum andcerebrospinal fluid.

LITERATURE CITED

1. Barbanti-Brodano, G., S. Oyanagi, M. Katz and H.Koprowski. 1969. Presence of two different viral agentsin brain cells of patients with subacute sclerosingpanencephalitis. Proc. Soc. Exp. Biol. Med.134:230-236.

2. Bather, R., J. M. Furesz, A. G. Fanok, S. D. Gill, and W.Yarosh. 1973. Long term infection of diploid Africangreen monkey brain cells by Schwarz measles vaccinevirus. J. Gen. Virol. 20:401-405.

3. Benda, P., J. Lightbody, G. Sato, L. Levine, and W.Sweet. 1968. Differentiated rat glial cell strain in tissueculture. Science 161:370.

4. Burnstein, T., L. J. Swango, and D. P. Byington. 1971.Nonspecific brain inhibitor against measles virus.Arch. Gesamte Virusforsch. 34:396-399.

5. Connolly, J. H., I. V. Allen, L. J. Hurwitz, and J. H. D.Miller. 1967. Measles-virus antibody and antigen insubacute sclerosing panencephalitis. Lancet 1:542-544.

6. Dayan, A. D., J. V. T. Gostling, J. L. Greaves, D. W.Stevens, and M. A. Woodhouse. 1967. Evidence of apseudomyxovirus in the brain in subacute sclerosingleucoencephalitis. Lancet 2:980-981.

7. Freeman, J. M., R. L. Magoffin, E. H. Lennette, and R.M. Herndon. 1967. Additional evidence of the relationbetween subacute inclusion-body encephalitis and

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measles virus. Lancet 2:129-131.8. Horta-Barbosa, L., D. A. Fuccillo, W. T. London, J. T.

Jabbour, W. Zeman, and J. L. Sever. 1969. Isolation ofmeasles virus from brain cell cultures of two patientswith subacute sclerosing panencephalitis. Proc. Soc.Exp. Biol. Med. 132:272-277.

9. Horta-Barbosa, L., D. A. Fucillo, and J. L. Sever. 1969.Subacute sclerosing panencephalitis: isolation ofmeasles virus from a brain biopsy. Nature (London)221:974.

10. Kashiwazaki, H., M. Homma, and N. Ishida. 1965. Assayof Sendai virus by immunofluorescence and hemad-sorbed cell-counting procedures. Proc. Soc. Exp. Biol.Med. 120:134-138.

11. Knight, P., R. Duff, and F. Rapp. 1972. Latency ofhuman measles virus in hamster cells. J. Virol.10:995-1001.

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12. Payne, F. E., J. V. Baublis, and H. H. Itabashi. 1969.Isolation of measles virus from cell cultures of brainfrom a patient with subacute sclerosing panencephali-tis. N. Engl. J. Med. 281:585-589.

13. Raine, C. R., L. A. Feldman, R. D. Sheppard, and M. R.Bornstein. 1971. Ultrastructural study of long-termmeasles infection in cultures of hamster dorsal-rootganglion. J. Virol. 8:318-329.

14. Reed, L. J., and H. A. Muench. 1938. A simple method fordetermining fifty percent endpoints. Am. J. Hyg.27:493-497.

15. Telez-Nagel, I., and D. H. Harter. 1966. Subacute scle-rosing panencephalitis and intracytoplasmic inclusions.Science 154:899-901.

16. Webb, H.E., S. J. Illavia, and G. D. Laurence. 1971.Measles-vaccine viruses in tissue culture of non-neuronal cells of human fetal brain. Lancet 2:4-5.

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