INFECTION AND IMMUNITY, Feb. 1977, p. 656-662Copyright © 1977 American Society for Microbiology

Vol. 15, No. 2Printed in U.S.A.

JC Virus, a Human Polyomavirus Associated withProgressive Multifocal Leukoencephalopathy: AdditionalBiological Characteristics and Antigenic Relationships

BILLIE L. PADGE¶T,* CHRISTINA M. ROGERS, AND DUARD L. WALKER

Department of Medical Microbiology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received for publication 23 August 1976

JC virus, a human polyomavirus, failed to grow or produce cytopathic effectsin any of a variety of cells tested other than primary human fetal glial (PHFG)cells. Cells tested included other primary human cells and glial cells from otheranimals. Only a rare cell in inoculated insusceptible human cell cultures pro-

duced T or virion antigen. In PHFG cell cultures JC virus produced subtlecytopathic effects, and the majority of progeny remained cell associated. Only afew cells in the heterogenous PHFG cell cultures contained T antigen at 24 hpostinoculation, and virion antigen was not detected until 48 h postinoculation.The infectivity ofJC virus was resistant to inactivation by ether and by heatingat 500 C for 1 h. A three-way minor antigenic relationship was demonstratedamong the virion antigens of JC virus, BK virus, and simian virus 40 byneutralization and/or hemagglutination inhibition tests. Serological evidence ispresented for the existence of JC virus as a distinct entity before the use ofsimian virus 40-contaminated poliovirus vaccines and for the nonexistence of ananimal reservoir for JC virus infection.

JC virus (JCV) was isolated from brain tis-sue from a patient with progressive multifocalleukocencephalopathy in 1971 (11). Subse-quently, this virus has been identified in braintissue from over 25 patients with this disease(2, 3, 4, 6, 8, 12, 18, 19; B. L. Padgett, unpub-lished data), and additional isolations havebeen made (3, 4, 6, 12, 19). The original isolateis the prototype of a new species of humanpapovavirus and, when distinction is neces-sary, should be designated as the MAD-1 strainof JCV.

In the initial description ofJCV, preliminarydata were presented indicating that it belongsin the polyomavirus genus of papovaviruses,and information developed subsequently con-cerning its nucleic acid (9) and oncogenicity (17)has been consistent with such a classification.One characteristic of JCV is its highly re-

stricted host cell range in vitro. It has beengrown only in primary human fetal glial(PHFG) cell cultures. This restriction has lim-ited virological studies of this human agent totwo or three laboratories worldwide. This re-port includes a more detailed description of theestablishment of PHFG cell cultures and of thegrowth and cytopathic effects (CPE) of JCV inthese cells. In addition, further characteristicsofJCV and its serological relationships to otherpapovaviruses are presented.

MATERIALS AND METHODS

Media. The initial growth medium for all glialcultures consisted of minimum essential medium(MEM) with L-glutamine and nonessential aminoacids (F-15; Grand Island Biological Company,Grand Island, N.Y.) and 10% fetal calf serum sup-plemented with 5 ml of 0.1 M HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid)/100 ml ofmedium (HMEM). Growth medium for establishedglial cultures and for all other primary human cellcultures consisted ofMEM and 10% fetal calf serum(MEM). Maintenance medium consisted of MEMand 3% fetal calf serum.

Cells. PHFG cell cultures were prepared fromfresh fragments of fetal brain tissue from 8- to 14-week-old abortuses. The meninges were removed ascompletely as possible with the aid of magnification.The tissue was broken up in HMEM by repeatedpipetting with a sterile capillary pipette followed byexpression through a 20-gauge hypodermic needle.The resulting suspension was distributed into 250-ml plastic flasks containing HMEM to give 9 ml of athick and coarse suspension containing approxi-mately 0.33 g of brain tissue. The flasks were incu-bated at 370C. Three days later unattached materialwas removed, pelleted, and resuspended in freshHMEM, and portions were redistributed among theflasks or put into new flasks if the original ones hadsufficient clumps of cells attached. This process wasrepeated every 3 days until no additional attach-ment of cells occurred. The cultures were changedinto MEM only when the cells were almost con-

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CHARACTERISTICS OF JC VIRUS 657

fluent. To obtain cultures that are rich in spongio-blasts, the following points are crucial: the tissuemust not be treated with trypsin, the tissue suspen-sion must be composed of coarse particles, and thecultures must be kept at a pH slightly below 7during attachment and initial growth. To preparesecondary PHFG cell cultures, the cells were re-moved from the plastic as follows. The cell sheet wascovered with saline A (14) containing 0.1% ethylene-diaminetetraacetate for 5 min, then 3 ml of saline Acontaining 0.05% trypsin and 0.02% ethylenedia-minetetraacetate was added. The cells detachedrapidly at ambient temperature and were pelletedfrom the trypsin solution as quickly as practical.

Other glial cell cultures, whether from adult orfetal tissues, were prepared similarly. Other pri-mary human fetal cell cultures were prepared fromorgans from 12- to 14-week-old abortuses. The or-gans were minced finely with scissors and trypsin-ized for 30 min in 0.25% trypsin. The resulting cellsuspensions were filtered through sterile gauze, pel-leted, resuspended in MEM, and put in appropriatecontainers. Primary African green monkey kidneycell cultures were purchased from Flow Laborato-ries, Rockville, Md. WI-38, CV-1, BSC-1, Vero, andChang conjunctiva cells were purchased from theAmerican Type Culture Collection, Rockville, Md.Mouse embryo cell cultures and L-809 cells wereobtained from June Osborn, University ofWisconsinMedical School, Madison, Wis. All cells were grownin the recommended medium.

Viruses. All experiments with JCV were per-formed with the original isolate that had been pas-saged between 9 and 12 times in PHFG cells. Pools ofJCV were prepared as described previously (9, 11).After being pelleted through 20% sucrose, the viruswas resuspended in MEM. A 5-ml pool preparedfrom 30 flasks of infected PHFG cells contained be-tween 5 x 105 and 2 x 106 hemagglutinating units(HAU) of virus. BK virus (seed virus obtained fromSylvia Gardner, Virus Reference Laboratory, Lon-don, England) was passaged in this laboratory inPHFG cultures that contained few spongioblasts.Pools ofBK virus (BKV) were prepared in the samemanner as those of JCV. SV40 strain 776 was pas-saged in CV-1 cells.

Titrations. Hemagglutination (HA) and hemag-glutination inhibition (HI) titrations were per-formed as described previously (10) except that amicrotiter assay (15) was used for HI titrations.Simian virus 40 (SV40) neutralization assays wereperformed in CV-1 cells. A plaque reduction assaywas used, and the neutralization titer is the recipro-cal of the serum dilution that reduced the number ofplaques to 50% of the control corrected to a controlcount of 200 plaques. The first JCV infectivity as-says were based on the observation of CPE in PHFGcell cultures, and titers are expressed as 50% tissueculture infectious dose (TCID50) as described previ-ously (10). Later, a fluorescent-cell assay was devel-oped. PHFG cells grown on 12-mm cover slips in 24-well plastic plates were inoculated with serial 10-fold dilutions of a virus suspension. Inoculated cul-tures were incubated for 9 days at 37C in an atmos-phere containing 5% CO2. The cover slips were fixed

in acetone for 10 min and stained for immunofluo-rescent counting with an anti-JCV serum obtainedfrom a rhesus monkey inoculated at birth with JCV.This serum contains antibodies against both JCVvirion and T antigens. The antigen-containing cellsper culture, two cultures per dilution, were countedat a magnification of 320 to provide a titer in fluores-cent-cell units. For JCV neutralization a fluorescentcell reduction assay was used, and the neutraliza-tion titer is the reciprocal of the serum dilution thatreduced the number of fluorescent cells to 50% of thecontrol corrected to a control value of 200 cells.

Immunofluorescent staining. All staining was bythe indirect technique using fluorescein-conjugatedantiglobulins obtained from Antibodies Inc., Davis,Calif. For detection of virion (V) antigen, cells werefixed in acetone for 15 min. The anti-JCV serumused was from hyperimmunized rabbits. For T-anti-gen detection, cells were fixed in acetone for 5 min.The antiserum used was a pool of sera from ham-sters bearing tumors induced by JCV. Brighter fluo-rescence was observed in the T-antigen system whencomplement was present in the initial staining step;therefore, the antiserum was diluted with an equalvolume of fresh guinea pig serum just before use.

RESULTS

Growth and CPE of JCV in PHFG cell cul-tures. PHFG cell cultures are heterogeneousbut two cell types predominate: large, pale-staining cells which will grow in a monolayer(astrocytes) and very small, round or bipolarcells with little cytoplasm (spongioblasts) (16).In our cultures, the spongioblasts grow in densemultilayered colonies that gradually spread outover the astrocytes (Fig. 1A). Under alkalineconditions spongioblasts appear to differentiateinto astrocytes.JCV produces CPE in both cell types with the

earliest changes occurring 10- to 14-days postin-oculation. Infected spongioblasts enlarge, andtheir shape changes from bipolar to epithelioid.Such subtle changes might go unnoticed in iso-lated, individual cells, but the altered appear-ance of entire spongioblast colonies can be de-tected easily (Fig. 1B). As reported previously(11), hematoxylin and eosin stain at this stageshowed many cells in the spongioblast coloniesin mitosis, some undergoing necrosis, andmany others with intranuclear inclusion bod-ies. Fuelgen or acridine orange staining dis-closed intranuclear bodies containing deoxyri-bonucleic acid (DNA). Later, the altered spon-gioblasts round up, shrink, and become ne-crotic; however, they may remain part of thecell sheet for an additional 2 to 3 weeks beforebreaking up completely.Although entire spongioblast colonies un-

dergo changes, only an occasional astrocyte ex-hibits CPE. Individual cells become greatly en-

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FIG. 1. PHFG cells in culture. (A) Uninoculated, control culture containing a dense colony of spongio-blasts; (B) another culture 21 days after inoculation with JCV. The two spongioblast colonies exhibit CPE.Cells are enlarged and have altered morphology. Hematoxylin and eosin stain. x102.

larged and their nucleus may be enlarged anddeeply staining, bizarre-shaped, or multinucle-ated. In maintenance medium the altered as-trocytes neither multiply nor become necroticduring 4 to 5 weeks of incubation.

Virion (V) antigens, detected by immuno-fluorescent staining, are found in both types ofcells (Fig. 2), and antigen is detected in manyastrocytes that do not show enlargement. How-ever, not all cells in the PHFG cell culturesappear to be susceptible to JCV because only 75to 90% of them contain V antigen 3 weeks afterinoculation.Progeny virus, as measured by HA, is associ-

ated strongly with the infected cells and celldebris. Only low levels of HA are ever detectedin the culture fluid and then only when muchdebris is present. JCV does not appear tospread readily from cell to cell except for thosewithin a spongioblast colony. If a low-titeredinoculum (10 HAU/250-ml flask) is added tocultures containing isolated spongioblast colo-nies, only some of the colonies will exhibit CPE5 weeks later. Also, in establishing the fluores-cent-cell assay of infectivity, no evidence of sec-ondary infection in the form of clusters of anti-gen-containing cells was seen until 10 or 11days postinoculation. Good yields of HA, 200-fold over input, are obtained only from PHFGcell cultures that are composed predominatelyof spongioblasts. To obtain the highest HAyields, it is necessary to process the infectedcultures as described in the preparation ofJCVpools (9, 11).

FIG. 2. JCV virion (V) antigen in nuclei ofPHFGcells 14 days after inoculation demonstrated by im-munofluorescence. x320.

Time of appearance of T and V antigens.Because JCV appeared to grow slowly in PHFGcell cultures, the time of appearance of T and Vantigens after infection of cells was deter-mined. For this experiment, replicate PHFGcell cultures were grown on 12-mm cover slipsand inoculated with 4 infectious units of JCV/cell. After an adsorption period, a potent JCV-

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VOL. 15, 1977

neutralizing antiserum was added to some ofthe cultures. At 24-h intervals for 5 days fourcultures were fixed in acetone for 10 min. Twocover slips were stained for T antigen and twowere stained for V antigen. The percentages ofcells containing T and V antigens were deter-mined by examining the cultures at a magnifi-cation of x320.At 24 h postinoculation less than 1% ofPHFG

cells contained detectable levels of T antigen(Table 1). The percentage of T antigen-contain-ing cells increased daily for 5 days even in thepresence of neutralizing antibody. Many astro-cytes contained T antigen; in fact, the firstpositive cells were astrocytes. V antigen ap-

peared later than T antigen. A few cells hadproduced V antigen at 48 h postinoculation, buteven at 5 days postinoculation, when close to60% of the cells contained T antigen, only ap-

proximately 2% contained V antigen. Althoughthe multiplicity of infection used was probablynot great enough to infect all of the cells, any

cell producing V antigen would be detected be-cause every cell was examined.Hemagglutinin. Pools ofJCV exhibit hemag-

glutinating activity that has been shown to be a

property of the infectious virion (9). JCV was

found to agglutinate human, guinea pig, andchicken erythrocytes, but not those of ham-sters, sheep, African green monkeys, or rhesusmonkeys. The erythrocytes are usable up to 7days after collection. The hemagglutinin was

not inactivated by heating at 56° C for 7 h, butit was destroyed when held at 80'C for 15 min.Host cell range of JCV. Our attempts to

TABLE 1. Time ofappearance ofT and V antigens inJCV-infected PHFG cells

Percentage of cells containing

Days after inoculation antigenT antigena V antigen

1 1 02 10 0.053 36 0.223 + antiserum' 30 0.174 46 1.05 57 2.35 + antiserum 59 NCd

a Percentage of T antigen-containing cells isbased on count of antigen-containing cells/100 cellsin each of five widely separated areas of the culture.

b The entire culture was examined and the V anti-gen-containing cells were counted. Percentages arethat number per average number of cells per culture(3 x 104).

e Medium containing antiserum sufficient to neu-tralize 106 fluorescent-cell units of JCV was addedafter adsorption on day zero and again on days 2 and4.

d Not counted.

CHARACTERISTICS OF JC VIRUS 659

cultivate JCV in cells other than PHFG cellshave failed. No CPE has been observed in un-stained cultures ofthe following cells after inoc-ulation with 100 HAU of JCV: primary humanembryonic kidney, lung, intestine, liver, andamnion; primary human adult testes; two hu-man diploid cells (L-809 and WI-38); a humanheteroploid cell (Chang conjunctiva); primaryAfrican green monkey kidney cells; BSC-1, CV-1, and Vero cells; adult rhesus monkey glialcells; hamster fetal glial cells; adult mink glialcells; mouse embryo cells. All of the inoculatedcell cultures were held for a minimum of 21days at 370C. At the end of the observationperiod all cultures were tested for HA activityand all were negative. For the following cells aserial blind passage was made: primary humanembryonic kidney, lung, intestine, liver, andamnion; WI-38 and Chang conjunctiva; primaryAfrican green monkey kidney, CV-1, and Vero;hamster fetal glial cells. These cultures werealso observed for 21 days, and again no CPE orHA was detected. In other experiments, cul-tures of WI-38, Vero, CV-1, and primary cul-tures of human embryonic kidney, lung, intes-tine, and liver cells were each inoculated with100 HAU of JCV and stained for JCV T and Vantigens 10 and 21 days later. At 10 days post-inoculation a few cells 'containing T and a fewcontaining V antigen were observed in all ofthe cultures of human cells, except primaryhuman embryonic lung cells, but no antigen-containing cells were seen in the Vero or CV-1cultures. The number of cells containing eitherantigen did not increase between 10 and 21days.

Stability of JCV. The sensitivity of JCV toethyl ether was determined by comparing theinfectivity titer of a sample of virus after expo-sure to ether with that of a sample not sotreated. Mixtures of equal volumes of virussuspension and either anesthetic ether or phos-phate-buffered saline (PBS) were held at 40Cfor 18 h with continuous agitation. After expo-sure to ether, the sample contained 104-5TCID50/ml as compared with 104 3 TCID5dml inthe control sample.For determination of heat stability, JCV was

pelleted by centrifugation at 96,900 x g for 2 h.The pellet was suspended in PBS and sonicallytreated briefly. Samples were diluted withequal volumes of PBS or phosphate-buffered 2M MgCl2 and heated at 500C for 1 h. The TCID50of the virus suspension both before and afterheating in PBS was 106/ml, but after heating in1 M MgCl2 it was less than 102/ml. Heat labilityin the presence of MgCl2 is a characteristic ofpolyomaviruses (7).

Serological relationships with other papo-vaviruses. Initially, using immunofluorescent

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660 PADGETT, ROGERS, AND WALKER

techniques, we found no serological relation-ship between the V antigen(s) ofJCV and otherpapovaviruses including the simian polyomavi-rus SV40 (11). Later, it was reported that cross-reactions did occur between JCV and both SV40and BKV, another human polyomavirus, whenhyperimmune sera were used in immune ag-glutination tests (1, 13). Therefore, we reexam-ined the serological relationship ofJCV to otherpapovaviruses and to SV40 and BKV in partic-ular, using antisera against V antigen(s) ofvarious papovaviruses. Since SV40 does nothemagglutinate, it was not possible to use thesimple and quick HI titration with all threeviruses. The HI test was used with BKV, neu-

tralization with SV40, and both tests were donewith JCV so that the titers obtained with JCVcould be compared directly with those obtainedwith the other two viruses. From the results(Table 2) it is obvious that JCV is a distinctvirus but one that does have a minor relation-ship to both SV40 and BKV. There was greatvariation among the antisera in the extent towhich they reacted with the heterologous vi-ruses; however, within each group of JCV,BKV, and SV40 antisera, one or more individ-ual antiserum(a) did react with the heterolo-gous viruses. Specifically, all of the BKV and

SV40 antisera tested neutralized JCV to some

extent although not all of them prevented HAby JCV. No reactions were observed betweenJCV and antisera against polyoma virus, Kvirus, or human papilloma virus.

Is JCV a "new virus"? Because of the weakantigenic relationship between JCV and thesimian polyomavirus SV40, the possibility thatJCV evolved from SV40 after the introductionof the latter virus into large numbers of hu-mans via contaminated lots of poliovirus vac-

cines was tested. Sera obtained locally beforethe introduction of poliovirus vaccines were ti-trated for HI antibodies against JCV. Forty-sixof 50 sera (92%) obtained from young adults in1950 and stored frozen had HI titers of 64 or

greater against JCV. Thus, infection of hu-mans with JCV was common before the intro-duction of poliovirus vaccines in 1954.Search for HI antibodies in sera from pri-

mates and other animals. Serological evidence(10) indicates that infection with JCV is com-

mon during childhood, but little else is knownconcerning the epidemiology of the infection.The possibility of an animal reservoir for JCVwas investigated by testing samples of sera

from a variety of animals for HI antibodiesagainst JCV. Sera tested included samples

TABLE 2. Serological relationship ofJCV to SV40, BKV, and other papovavirusesTest virus

Serum against: SV40 JCV BKV

Neut. titera Neut. titerb HI tite' HI titer

JCV (rabbit 283) 5 2.0 x 106 5.1 x 105 512JCV (rabbit 280) <10 3.4 x 105 6.4 x 104 <8JCV (M-PML: rabbit 2)d NDe 1.7 x 104 5.1 X 103 <8BKV (rabbit 288) ND 27 <8 1 X 103BKV (rabbit)' 250 60 32 1.3 x 105SV40 (calf)0 6 x 103 36 <8 <8SV40 (horse)0 2.5 x 104 2.6 x 103 1 X 103 <8SV40 (rabbit 4)d 9 X 104 195 <8 128SV40PML.2 (rabbit 4: 1973)d 1.3 x 104 190 16 <8SV40PML.I (rabbit: 1971)d 1.3 x 104 5.1 x 104 4.1 x 103 512SV40PML.I (rabbit 2: 1973)d 2.5 x 104 60 32 256Polyomavirush ND <8 <32 <8K virus' ND <8 8 NDHuman papilloma virus ND <8 8 <8

a Neutralization (neut.) titer is the reciprocal of the serum dilution that reduced the number ofplaques to50% of the control (control = 200 plaques).

b Neutralization titer is the reciprocal ofthe serum dilution that reduced the number offluorescent cells to50% of the control (control = 200 fluorescent cells).

e HI titer is the reciprocal of the highest serum dilution that completely prevented HA.d Serum provided by 0. Narayan and L. Weiner, Johns Hopkins University, Baltimore, Md.e ND, Not done.f Serum provided by G. di Mayorca, University of Illinois, Chicago, Ill.D Serum purchased from Flow Laboratories, Inc., Rockville, Md.h Serum purchased from Microbiological Associates, Bethesda, Md.I Serum provided by K. Takemoto, National Institute ofAllergy and Infectious Diseases, Bethesda, Md.

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CHARACTERISTICS OF JC VIRUS 661

from a variety of primates and animals fre-quently associated with humans as pets. Allsera were treated with NaIO4 before use, a tech-nique that successfully removed nonspecific in-hibitors of JCV HA from human sera (10).Many of the animal sera so treated had HItiters greater than 32 and, therefore, would beconsidered positive (Table 3). To test the speci-ficity of the inhibition, a sample of all suchpositivev" sera was extracted with acetone (5)and retitrated. Except for sera from threeguinea pigs, the HI titers were now all lessthan 20. The guinea pig sera were tested forneutralizing activity against JCV and all threewere negative. Thus none of the animal seratested had antibodies against JCV.

DISCUSSIONThis report extends the characterization of

JCV, a recently discovered human papovavi-rus. With the demonstration of resistance toinactivation by heat and ether, JCV has now

been shown to have all the characteristics com-

mon to members of the polyomavirus genus ofpapovaviruses. Moreover, the established anti-genic relationship between JCV, BKV, andSV40 indicates that these primate viruses forma subgroup within the polyomavirus genus.However, the serological evidence presentedhere clearly shows that JCV did not evolvefrom SV40 in the recent past.

TABLE 3. Test for HI antibodies against JCV insera of various animals

Animal No. tested Range of HItitersa

Squirrel monkey" 5 <8Cebus monkey" 5 <8Owl monkey 41 <8-16Rhesus monkey" 17 <8-32Marmosetd 32 <8-32Cat 25 <8Dog 21 <8-256Domestic rabbit 20 <8-32Guinea pig 28 <8-128Syrian hamster 40 <8-512Swiss mousee 9 512-1024Mink 21 <8-32

a HI titers are expressed as the reciprocal of thehighest serum dilution that inhibited hemaggluti-nation completely.

b Sera provided by H. Rabin, Litton-Bionetics Re-search Laboratories, Kensington, Md.

e Sera provided by W. London, National Instituteof Neurological and Communicative Disorders andStroke, Bethesda, Md.

d Sera provided by L. Wolfe, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Ill.

e Each serum was a pool of sera from at least 25animals.

With many of the hyperimmune sera, cross-reactions were observed by either neutraliza-tion of HI among the virion antigens of JCV,BKV, and SV40. Antisera produced in rabbitsusually gave much lower heterologous titersthan homologous titers, indicating that the an-tigenic relationships are of a minor nature. Theexception was an antiserum against early pas-sage SV40PMII virus which gave very high neu-tralization and HI titers with JCV. This indica-tion of a close relationship between JCV andSV40PMLI virus was not confirmed using an-other antiserum against the latter virus or us-ing an antiserum against SV40PML2 virus. TheSV40PMLI and SV40PML2 viruses are SV40strains isolated from two patients with progres-sive multifocal leukoencephalopathy (18). Theyare serologically indistinguishable from eachother and from a standard laboratory strain ofSV40. We have no explanation for the differentresults obtained consistently with the twoSV40PMLI antisera.Polyomaviruses in general have restricted

host ranges in vivo, and we found no antibodiesagainst JCV in sera from a variety of animalsincluding several species of primates, commonpets, and animals bred and raised for labora-tory use. These results suggest that JCV doesnot naturally infect any of these animals. Serafrom several species, however, did contain non-specific inhibitors of JCV HA which were notremoved by treatment with NaIO4. This findingemphasizes the necessity to confirm the speci-ficity of observed HI reactions whenever serafrom species not previously tested are used.JCV exhibits an even greater restriction in

its host cell range in vitro. Although manydifferent human cell cultures have been tested,growth ofJCV was obtained only in PHFG cellcultures. Indeed, JCV did not even produce Tantigen in most of the other cells. IfJCV pene-trates these insusceptible cells, the infection isaborted at a very early stage in replication. It isreasonable to assume that JCV grows in addi-tional cell types in its host. After all, JCV is aubiquitous virus and infection with it is a com-mon childhood event. Its limitation to PHFGcells in vitro might be due simply to one ormore of the differences known to exist betweencells in situ and cells in culture. However, inpatients with progressive multifocal leukoen-cephalopathy, JCV is multiplying in certainglial cells, and the virus used in these experi-ments was isolated from an infected brain. It ispossible that the observed restriction to glialcells in vitro is the result of some change in thevirus brought about by its prior growth in suchcells in vivo.The requirement for primary cultures con-

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662 PADGETT, ROGERS, AND WALKER

training a high proportion of spongioblasts hasseverely hampered study of JCV. Fetal braintissue is difficult to obtain on a regular basis,and even with careful handling, there is greatvariation in the composition of the cultures,with many batches containing insufficientspongioblasts to be useful. The difficulties pre-sented by the cell system would be compensatedsomewhat if the virus was cytolytic, multipliedrapidly, and was released into the fluid effi-ciently, but JCV has just the opposite proper-ties. The combination of this virus and cellsystem, therefore, has made it impossible toplaque purify JCV or even to produce usefulquantities of virus from limit-dilution passage.It is manifestly impossible to perform molecu-lar-biological experiments of the nicety obtain-able with SV40 or polyomavirus. Even simplegrowth curve experiments will be crude. Thebeginnings of such a study, reported here astime of appearance of T and V antigens, indi-cates that the growth cycle ofJCV is protractedand obviously asynchronous. The observedslowness may be due in part to the presence ofdefective virions in the JCV pool because DNAextracted from similar pools was found to beheterogeneous with respect to size ofDNA mol-ecules (9).

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant

no. AI-11217 from the National Institute of Allergy andInfectious Diseases.

LITERATURE CITED

1. Albert, A. E., and G. M. ZuRhein. 1974. Application ofimmune electron microscopy to the study of the anti-genic relationships between three new human papo-vaviruses. Int. Arch. Allergy Appl. Immunol. 46:405-416.

2. Bauer, W. R., A. P. Turel, Jr., and K. P. Johnson. 1973.Progressive multifocal leukoencephalopathy and cy-tarabine. J. Am. Med. Assoc. 226:174-176.

3. Cathala, F., J.-J. Hauw, and R. Escourolle. 1973.Isolement et etude ultrastructurale d'un virus de typepapova non neutralist par le serum de reference anti-SV40 au cours de la leucoencdphalopathie multifocaleprogressive. C. R. Acad. Sci. 276:1081-1084.

4. Field, A. M., S. D. Gardner, R. A. Goodbody, and M. A.Woodhouse. 1974. Identity of a newly isolated humanpolyomavirus from a patient with progressive multi-focal leucoencephalopathy. J. Clin. Pathol. 27:341-

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347.5. Hammon, W. M., and G. E. Sather. 1969. Arboviruses,

p. 227-280. In E. H. Lennette and N. J. Schmidt (ed.),Diagnostic procedures for viral and rickettsial infec-tions, 4th ed. American Public Health Association,Inc., New York.

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