INFECTION AND IMMUNITY, Feb. 1977, p. 656-662 Copyright © 1977 American Society for Microbiology
Vol. 15, No. 2 Printed in U.S.A.
JC Virus, a Human Polyomavirus Associated with Progressive Multifocal Leukoencephalopathy: Additional Biological 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 effects in 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 other animals. Only a rare cell in inoculated insusceptible human cell cultures produced T or virion antigen. In PHFG cell cultures JC virus produced subtle cytopathic effects, and the majority of progeny remained cell associated. Only a few cells in the heterogenous PHFG cell cultures contained T antigen at 24 h postinoculation, and virion antigen was not detected until 48 h postinoculation. The infectivity of JC virus was resistant to inactivation by ether and by heating at 500 C for 1 h. A three-way minor antigenic relationship was demonstrated among the virion antigens of JC virus, BK virus, and simian virus 40 by neutralization and/or hemagglutination inhibition tests. Serological evidence is presented for the existence of JC virus as a distinct entity before the use of simian virus 40-contaminated poliovirus vaccines and for the nonexistence of an animal reservoir for JC virus infection. MATERIALS AND METHODS
JC virus (JCV) was isolated from brain tissue from a patient with progressive multifocal leukocencephalopathy in 1971 (11). Subsequently, this virus has been identified in brain tissue from over 25 patients with this disease (2, 3, 4, 6, 8, 12, 18, 19; B. L. Padgett, unpublished data), and additional isolations have been made (3, 4, 6, 12, 19). The original isolate is the prototype of a new species of human papovavirus and, when distinction is necessary, should be designated as the MAD-1 strain of JCV. In the initial description of JCV, preliminary data were presented indicating that it belongs in the polyomavirus genus of papovaviruses, and information developed subsequently concerning its nucleic acid (9) and oncogenicity (17) has been consistent with such a classification. One characteristic of JCV is its highly restricted host cell range in vitro. It has been grown only in primary human fetal glial (PHFG) cell cultures. This restriction has limited virological studies of this human agent to two or three laboratories worldwide. This report includes a more detailed description of the establishment of PHFG cell cultures and of the growth and cytopathic effects (CPE) of JCV in these cells. In addition, further characteristics of JCV and its serological relationships to other papovaviruses are presented.
Media. The initial growth medium for all glial cultures consisted of minimum essential medium (MEM) with L-glutamine and nonessential amino acids (F-15; Grand Island Biological Company, Grand Island, N.Y.) and 10% fetal calf serum supplemented with 5 ml of 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)/100 ml of medium (HMEM). Growth medium for established glial cultures and for all other primary human cell cultures consisted of MEM and 10% fetal calf serum (MEM). Maintenance medium consisted of MEM and 3% fetal calf serum. Cells. PHFG cell cultures were prepared from fresh fragments of fetal brain tissue from 8- to 14week-old abortuses. The meninges were removed as completely as possible with the aid of magnification. The tissue was broken up in HMEM by repeated pipetting with a sterile capillary pipette followed by expression through a 20-gauge hypodermic needle. The resulting suspension was distributed into 250ml plastic flasks containing HMEM to give 9 ml of a thick and coarse suspension containing approximately 0.33 g of brain tissue. The flasks were incubated at 370C. Three days later unattached material was removed, pelleted, and resuspended in fresh HMEM, and portions were redistributed among the flasks or put into new flasks if the original ones had sufficient clumps of cells attached. This process was repeated every 3 days until no additional attachment of cells occurred. The cultures were changed into MEM only when the cells were almost con656
VOL. 15, 1977 fluent. To obtain cultures that are rich in spongioblasts, the following points are crucial: the tissue must not be treated with trypsin, the tissue suspension must be composed of coarse particles, and the cultures must be kept at a pH slightly below 7 during attachment and initial growth. To prepare secondary PHFG cell cultures, the cells were removed from the plastic as follows. The cell sheet was covered with saline A (14) containing 0.1% ethylenediaminetetraacetate for 5 min, then 3 ml of saline A containing 0.05% trypsin and 0.02% ethylenediaminetetraacetate was added. The cells detached rapidly at ambient temperature and were pelleted from the trypsin solution as quickly as practical. Other glial cell cultures, whether from adult or fetal tissues, were prepared similarly. Other primary human fetal cell cultures were prepared from organs from 12- to 14-week-old abortuses. The organs were minced finely with scissors and trypsinized for 30 min in 0.25% trypsin. The resulting cell suspensions were filtered through sterile gauze, pelleted, resuspended in MEM, and put in appropriate containers. Primary African green monkey kidney cell cultures were purchased from Flow Laboratories, Rockville, Md. WI-38, CV-1, BSC-1, Vero, and Chang conjunctiva cells were purchased from the American Type Culture Collection, Rockville, Md. Mouse embryo cell cultures and L-809 cells were obtained from June Osborn, University of Wisconsin Medical School, Madison, Wis. All cells were grown in the recommended medium. Viruses. All experiments with JCV were performed with the original isolate that had been passaged between 9 and 12 times in PHFG cells. Pools of JCV were prepared as described previously (9, 11). After being pelleted through 20% sucrose, the virus was resuspended in MEM. A 5-ml pool prepared from 30 flasks of infected PHFG cells contained between 5 x 105 and 2 x 106 hemagglutinating units (HAU) of virus. BK virus (seed virus obtained from Sylvia Gardner, Virus Reference Laboratory, London, England) was passaged in this laboratory in PHFG cultures that contained few spongioblasts. Pools of BK virus (BKV) were prepared in the same manner as those of JCV. SV40 strain 776 was passaged in CV-1 cells. Titrations. Hemagglutination (HA) and hemagglutination inhibition (HI) titrations were performed as described previously (10) except that a microtiter assay (15) was used for HI titrations. Simian virus 40 (SV40) neutralization assays were performed in CV-1 cells. A plaque reduction assay was used, and the neutralization titer is the reciprocal of the serum dilution that reduced the number of plaques to 50% of the control corrected to a control count of 200 plaques. The first JCV infectivity assays were based on the observation of CPE in PHFG cell cultures, and titers are expressed as 50% tissue culture infectious dose (TCID50) as described previously (10). Later, a fluorescent-cell assay was developed. PHFG cells grown on 12-mm cover slips in 24well plastic plates were inoculated with serial 10fold dilutions of a virus suspension. Inoculated cultures were incubated for 9 days at 37C in an atmosphere containing 5% CO2. The cover slips were fixed
CHARACTERISTICS OF JC VIRUS
657
in acetone for 10 min and stained for immunofluorescent counting with an anti-JCV serum obtained from a rhesus monkey inoculated at birth with JCV. This serum contains antibodies against both JCV virion and T antigens. The antigen-containing cells per culture, two cultures per dilution, were counted at a magnification of 320 to provide a titer in fluorescent-cell units. For JCV neutralization a fluorescent cell reduction assay was used, and the neutralization titer is the reciprocal of the serum dilution that reduced the number of fluorescent cells to 50% of the control corrected to a control value of 200 cells. Immunofluorescent staining. All staining was by the indirect technique using fluorescein-conjugated antiglobulins obtained from Antibodies Inc., Davis, Calif. For detection of virion (V) antigen, cells were fixed in acetone for 15 min. The anti-JCV serum used was from hyperimmunized rabbits. For T-antigen detection, cells were fixed in acetone for 5 min. The antiserum used was a pool of sera from hamsters bearing tumors induced by JCV. Brighter fluorescence was observed in the T-antigen system when complement was present in the initial staining step; therefore, the antiserum was diluted with an equal volume of fresh guinea pig serum just before use.
RESULTS Growth and CPE of JCV in PHFG cell cultures. PHFG cell cultures are heterogeneous but two cell types predominate: large, palestaining cells which will grow in a monolayer (astrocytes) and very small, round or bipolar cells with little cytoplasm (spongioblasts) (16). In our cultures, the spongioblasts grow in dense multilayered colonies that gradually spread out over the astrocytes (Fig. 1A). Under alkaline conditions spongioblasts appear to differentiate into astrocytes. JCV produces CPE in both cell types with the earliest changes occurring 10- to 14-days postinoculation. Infected spongioblasts enlarge, and their shape changes from bipolar to epithelioid. Such subtle changes might go unnoticed in isolated, individual cells, but the altered appearance of entire spongioblast colonies can be detected easily (Fig. 1B). As reported previously (11), hematoxylin and eosin stain at this stage showed many cells in the spongioblast colonies in mitosis, some undergoing necrosis, and many others with intranuclear inclusion bodies. Fuelgen or acridine orange staining disclosed intranuclear bodies containing deoxyribonucleic acid (DNA). Later, the altered spongioblasts round up, shrink, and become necrotic; however, they may remain part of the cell sheet for an additional 2 to 3 weeks before breaking up completely. Although entire spongioblast colonies undergo changes, only an occasional astrocyte exhibits CPE. Individual cells become greatly en-
658
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AND WALKER
INFECT. IMMUN.
FIG. 1. PHFG cells in culture. (A) Uninoculated, control culture containing a dense colony of spongioblasts; (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 and deeply staining, bizarre-shaped, or multinucleated. In maintenance medium the altered astrocytes neither multiply nor become necrotic during 4 to 5 weeks of incubation. Virion (V) antigens, detected by immunofluorescent staining, are found in both types of cells (Fig. 2), and antigen is detected in many astrocytes that do not show enlargement. However, not all cells in the PHFG cell cultures appear to be susceptible to JCV because only 75 to 90% of them contain V antigen 3 weeks after inoculation. Progeny virus, as measured by HA, is associated strongly with the infected cells and cell debris. Only low levels of HA are ever detected in the culture fluid and then only when much debris is present. JCV does not appear to spread readily from cell to cell except for those within a spongioblast colony. If a low-titered inoculum (10 HAU/250-ml flask) is added to cultures containing isolated spongioblast colonies, only some of the colonies will exhibit CPE 5 weeks later. Also, in establishing the fluorescent-cell assay of infectivity, no evidence of secondary infection in the form of clusters of antigen-containing cells was seen until 10 or 11 days postinoculation. Good yields of HA, 200fold over input, are obtained only from PHFG cell cultures that are composed predominately of spongioblasts. To obtain the highest HA yields, it is necessary to process the infected cultures as described in the preparation of JCV pools (9, 11).
FIG. 2. JCV virion (V) antigen in nuclei of PHFG cells 14 days after inoculation demonstrated by immunofluorescence. x320.
Time of appearance of T and V antigens. Because JCV appeared to grow slowly in PHFG cell cultures, the time of appearance of T and V antigens after infection of cells was determined. For this experiment, replicate PHFG cell cultures were grown on 12-mm cover slips and inoculated with 4 infectious units of JCV/ cell. After an adsorption period, a potent JCV-
CHARACTERISTICS OF JC VIRUS
VOL. 15, 1977
neutralizing antiserum was added to some of the cultures. At 24-h intervals for 5 days four cultures were fixed in acetone for 10 min. Two cover slips were stained for T antigen and two were stained for V antigen. The percentages of cells containing T and V antigens were determined by examining the cultures at a magnification of x320. At 24 h postinoculation less than 1% of PHFG cells contained detectable levels of T antigen (Table 1). The percentage of T antigen-containing cells increased daily for 5 days even in the presence of neutralizing antibody. Many astrocytes contained T antigen; in fact, the first positive cells were astrocytes. V antigen appeared later than T antigen. A few cells had produced V antigen at 48 h postinoculation, but even at 5 days postinoculation, when close to 60% of the cells contained T antigen, only approximately 2% contained V antigen. Although the multiplicity of infection used was probably not great enough to infect all of the cells, any cell producing V antigen would be detected because every cell was examined. Hemagglutinin. Pools ofJCV exhibit hemagglutinating activity that has been shown to be a property of the infectious virion (9). JCV was found to agglutinate human, guinea pig, and chicken erythrocytes, but not those of hamsters, sheep, African green monkeys, or rhesus monkeys. The erythrocytes are usable up to 7 days after collection. The hemagglutinin was not inactivated by heating at 56° C for 7 h, but it was destroyed when held at 80'C for 15 min. Host cell range of JCV. Our attempts to TABLE 1. Time of appearance of T and V antigens in JCV-infected PHFG cells Percentage of cells containing
antigen
Days after inoculation T antigena 1
V
antigen
1 0 0.05 2 10 36 0.22 3 30 0.17 3 + antiserum' 4 46 1.0 57 2.3 5 NCd 5 + antiserum 59 a Percentage of T antigen-containing cells is based on count of antigen-containing cells/100 cells in each of five widely separated areas of the culture. b The entire culture was examined and the V antigen-containing cells were counted. Percentages are that number per average number of cells per culture (3 x 104). e Medium containing antiserum sufficient to neutralize 106 fluorescent-cell units of JCV was added after adsorption on day zero and again on days 2 and
4. d
Not counted.
659
cultivate JCV in cells other than PHFG cells have failed. No CPE has been observed in unstained cultures of the following cells after inoculation with 100 HAU of JCV: primary human embryonic kidney, lung, intestine, liver, and amnion; primary human adult testes; two human diploid cells (L-809 and WI-38); a human heteroploid cell (Chang conjunctiva); primary African green monkey kidney cells; BSC-1, CV1, and Vero cells; adult rhesus monkey glial cells; hamster fetal glial cells; adult mink glial cells; mouse embryo cells. All of the inoculated cell cultures were held for a minimum of 21 days at 370C. At the end of the observation period all cultures were tested for HA activity and all were negative. For the following cells a serial blind passage was made: primary human embryonic kidney, lung, intestine, liver, and amnion; WI-38 and Chang conjunctiva; primary African green monkey kidney, CV-1, and Vero; hamster fetal glial cells. These cultures were also observed for 21 days, and again no CPE or HA was detected. In other experiments, cultures of WI-38, Vero, CV-1, and primary cultures of human embryonic kidney, lung, intestine, and liver cells were each inoculated with 100 HAU of JCV and stained for JCV T and V antigens 10 and 21 days later. At 10 days postinoculation a few cells 'containing T and a few containing V antigen were observed in all of the cultures of human cells, except primary human embryonic lung cells, but no antigencontaining cells were seen in the Vero or CV-1 cultures. The number of cells containing either antigen did not increase between 10 and 21 days. Stability of JCV. The sensitivity of JCV to ethyl ether was determined by comparing the infectivity titer of a sample of virus after exposure to ether with that of a sample not so treated. Mixtures of equal volumes of virus suspension and either anesthetic ether or phosphate-buffered saline (PBS) were held at 40C for 18 h with continuous agitation. After exposure to ether, the sample contained 104-5 TCID50/ml as compared with 104 3 TCID5dml in the 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 sonically treated briefly. Samples were diluted with equal volumes of PBS or phosphate-buffered 2 M MgCl2 and heated at 500C for 1 h. The TCID50 of the virus suspension both before and after heating in PBS was 106/ml, but after heating in 1 M MgCl2 it was less than 102/ml. Heat lability in the presence of MgCl2 is a characteristic of polyomaviruses (7). Serological relationships with other papovaviruses. Initially, using immunofluorescent
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techniques, we found no serological relationship between the V antigen(s) of JCV and other papovaviruses including the simian polyomavirus SV40 (11). Later, it was reported that crossreactions did occur between JCV and both SV40 and BKV, another human polyomavirus, when hyperimmune sera were used in immune agglutination tests (1, 13). Therefore, we reexamined the serological relationship of JCV to other papovaviruses and to SV40 and BKV in particular, using antisera against V antigen(s) of various papovaviruses. Since SV40 does not hemagglutinate, it was not possible to use the simple and quick HI titration with all three viruses. The HI test was used with BKV, neutralization with SV40, and both tests were done with JCV so that the titers obtained with JCV could be compared directly with those obtained with the other two viruses. From the results (Table 2) it is obvious that JCV is a distinct virus but one that does have a minor relationship to both SV40 and BKV. There was great variation among the antisera in the extent to which they reacted with the heterologous viruses; however, within each group of JCV, BKV, and SV40 antisera, one or more individual antiserum(a) did react with the heterologous viruses. Specifically, all of the BKV and
INFECT. IMMUN.
SV40 antisera tested neutralized JCV to some extent although not all of them prevented HA by JCV. No reactions were observed between JCV and antisera against polyoma virus, K virus, or human papilloma virus. Is JCV a "new virus"? Because of the weak antigenic relationship between JCV and the simian polyomavirus SV40, the possibility that JCV evolved from SV40 after the introduction of the latter virus into large numbers of humans via contaminated lots of poliovirus vaccines was tested. Sera obtained locally before the introduction of poliovirus vaccines were titrated for HI antibodies against JCV. Forty-six of 50 sera (92%) obtained from young adults in 1950 and stored frozen had HI titers of 64 or greater against JCV. Thus, infection of humans with JCV was common before the introduction of poliovirus vaccines in 1954. Search for HI antibodies in sera from primates and other animals. Serological evidence (10) indicates that infection with JCV is common during childhood, but little else is known concerning the epidemiology of the infection. The possibility of an animal reservoir for JCV was investigated by testing samples of sera from a variety of animals for HI antibodies against JCV. Sera tested included samples
TABLE 2. Serological relationship of JCV to SV40, BKV, and other papovaviruses Test virus
Serum against:
JCV
SV40
BKV
HI tite' HI titer 5.1 x 105 2.0 x 106 512 3.4 x 105 6.4 x 104
Vol. 15, No. 2 Printed in U.S.A.
JC Virus, a Human Polyomavirus Associated with Progressive Multifocal Leukoencephalopathy: Additional Biological 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 effects in 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 other animals. Only a rare cell in inoculated insusceptible human cell cultures produced T or virion antigen. In PHFG cell cultures JC virus produced subtle cytopathic effects, and the majority of progeny remained cell associated. Only a few cells in the heterogenous PHFG cell cultures contained T antigen at 24 h postinoculation, and virion antigen was not detected until 48 h postinoculation. The infectivity of JC virus was resistant to inactivation by ether and by heating at 500 C for 1 h. A three-way minor antigenic relationship was demonstrated among the virion antigens of JC virus, BK virus, and simian virus 40 by neutralization and/or hemagglutination inhibition tests. Serological evidence is presented for the existence of JC virus as a distinct entity before the use of simian virus 40-contaminated poliovirus vaccines and for the nonexistence of an animal reservoir for JC virus infection. MATERIALS AND METHODS
JC virus (JCV) was isolated from brain tissue from a patient with progressive multifocal leukocencephalopathy in 1971 (11). Subsequently, this virus has been identified in brain tissue from over 25 patients with this disease (2, 3, 4, 6, 8, 12, 18, 19; B. L. Padgett, unpublished data), and additional isolations have been made (3, 4, 6, 12, 19). The original isolate is the prototype of a new species of human papovavirus and, when distinction is necessary, should be designated as the MAD-1 strain of JCV. In the initial description of JCV, preliminary data were presented indicating that it belongs in the polyomavirus genus of papovaviruses, and information developed subsequently concerning its nucleic acid (9) and oncogenicity (17) has been consistent with such a classification. One characteristic of JCV is its highly restricted host cell range in vitro. It has been grown only in primary human fetal glial (PHFG) cell cultures. This restriction has limited virological studies of this human agent to two or three laboratories worldwide. This report includes a more detailed description of the establishment of PHFG cell cultures and of the growth and cytopathic effects (CPE) of JCV in these cells. In addition, further characteristics of JCV and its serological relationships to other papovaviruses are presented.
Media. The initial growth medium for all glial cultures consisted of minimum essential medium (MEM) with L-glutamine and nonessential amino acids (F-15; Grand Island Biological Company, Grand Island, N.Y.) and 10% fetal calf serum supplemented with 5 ml of 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)/100 ml of medium (HMEM). Growth medium for established glial cultures and for all other primary human cell cultures consisted of MEM and 10% fetal calf serum (MEM). Maintenance medium consisted of MEM and 3% fetal calf serum. Cells. PHFG cell cultures were prepared from fresh fragments of fetal brain tissue from 8- to 14week-old abortuses. The meninges were removed as completely as possible with the aid of magnification. The tissue was broken up in HMEM by repeated pipetting with a sterile capillary pipette followed by expression through a 20-gauge hypodermic needle. The resulting suspension was distributed into 250ml plastic flasks containing HMEM to give 9 ml of a thick and coarse suspension containing approximately 0.33 g of brain tissue. The flasks were incubated at 370C. Three days later unattached material was removed, pelleted, and resuspended in fresh HMEM, and portions were redistributed among the flasks or put into new flasks if the original ones had sufficient clumps of cells attached. This process was repeated every 3 days until no additional attachment of cells occurred. The cultures were changed into MEM only when the cells were almost con656
VOL. 15, 1977 fluent. To obtain cultures that are rich in spongioblasts, the following points are crucial: the tissue must not be treated with trypsin, the tissue suspension must be composed of coarse particles, and the cultures must be kept at a pH slightly below 7 during attachment and initial growth. To prepare secondary PHFG cell cultures, the cells were removed from the plastic as follows. The cell sheet was covered with saline A (14) containing 0.1% ethylenediaminetetraacetate for 5 min, then 3 ml of saline A containing 0.05% trypsin and 0.02% ethylenediaminetetraacetate was added. The cells detached rapidly at ambient temperature and were pelleted from the trypsin solution as quickly as practical. Other glial cell cultures, whether from adult or fetal tissues, were prepared similarly. Other primary human fetal cell cultures were prepared from organs from 12- to 14-week-old abortuses. The organs were minced finely with scissors and trypsinized for 30 min in 0.25% trypsin. The resulting cell suspensions were filtered through sterile gauze, pelleted, resuspended in MEM, and put in appropriate containers. Primary African green monkey kidney cell cultures were purchased from Flow Laboratories, Rockville, Md. WI-38, CV-1, BSC-1, Vero, and Chang conjunctiva cells were purchased from the American Type Culture Collection, Rockville, Md. Mouse embryo cell cultures and L-809 cells were obtained from June Osborn, University of Wisconsin Medical School, Madison, Wis. All cells were grown in the recommended medium. Viruses. All experiments with JCV were performed with the original isolate that had been passaged between 9 and 12 times in PHFG cells. Pools of JCV were prepared as described previously (9, 11). After being pelleted through 20% sucrose, the virus was resuspended in MEM. A 5-ml pool prepared from 30 flasks of infected PHFG cells contained between 5 x 105 and 2 x 106 hemagglutinating units (HAU) of virus. BK virus (seed virus obtained from Sylvia Gardner, Virus Reference Laboratory, London, England) was passaged in this laboratory in PHFG cultures that contained few spongioblasts. Pools of BK virus (BKV) were prepared in the same manner as those of JCV. SV40 strain 776 was passaged in CV-1 cells. Titrations. Hemagglutination (HA) and hemagglutination inhibition (HI) titrations were performed as described previously (10) except that a microtiter assay (15) was used for HI titrations. Simian virus 40 (SV40) neutralization assays were performed in CV-1 cells. A plaque reduction assay was used, and the neutralization titer is the reciprocal of the serum dilution that reduced the number of plaques to 50% of the control corrected to a control count of 200 plaques. The first JCV infectivity assays were based on the observation of CPE in PHFG cell cultures, and titers are expressed as 50% tissue culture infectious dose (TCID50) as described previously (10). Later, a fluorescent-cell assay was developed. PHFG cells grown on 12-mm cover slips in 24well plastic plates were inoculated with serial 10fold dilutions of a virus suspension. Inoculated cultures were incubated for 9 days at 37C in an atmosphere containing 5% CO2. The cover slips were fixed
CHARACTERISTICS OF JC VIRUS
657
in acetone for 10 min and stained for immunofluorescent counting with an anti-JCV serum obtained from a rhesus monkey inoculated at birth with JCV. This serum contains antibodies against both JCV virion and T antigens. The antigen-containing cells per culture, two cultures per dilution, were counted at a magnification of 320 to provide a titer in fluorescent-cell units. For JCV neutralization a fluorescent cell reduction assay was used, and the neutralization titer is the reciprocal of the serum dilution that reduced the number of fluorescent cells to 50% of the control corrected to a control value of 200 cells. Immunofluorescent staining. All staining was by the indirect technique using fluorescein-conjugated antiglobulins obtained from Antibodies Inc., Davis, Calif. For detection of virion (V) antigen, cells were fixed in acetone for 15 min. The anti-JCV serum used was from hyperimmunized rabbits. For T-antigen detection, cells were fixed in acetone for 5 min. The antiserum used was a pool of sera from hamsters bearing tumors induced by JCV. Brighter fluorescence was observed in the T-antigen system when complement was present in the initial staining step; therefore, the antiserum was diluted with an equal volume of fresh guinea pig serum just before use.
RESULTS Growth and CPE of JCV in PHFG cell cultures. PHFG cell cultures are heterogeneous but two cell types predominate: large, palestaining cells which will grow in a monolayer (astrocytes) and very small, round or bipolar cells with little cytoplasm (spongioblasts) (16). In our cultures, the spongioblasts grow in dense multilayered colonies that gradually spread out over the astrocytes (Fig. 1A). Under alkaline conditions spongioblasts appear to differentiate into astrocytes. JCV produces CPE in both cell types with the earliest changes occurring 10- to 14-days postinoculation. Infected spongioblasts enlarge, and their shape changes from bipolar to epithelioid. Such subtle changes might go unnoticed in isolated, individual cells, but the altered appearance of entire spongioblast colonies can be detected easily (Fig. 1B). As reported previously (11), hematoxylin and eosin stain at this stage showed many cells in the spongioblast colonies in mitosis, some undergoing necrosis, and many others with intranuclear inclusion bodies. Fuelgen or acridine orange staining disclosed intranuclear bodies containing deoxyribonucleic acid (DNA). Later, the altered spongioblasts round up, shrink, and become necrotic; however, they may remain part of the cell sheet for an additional 2 to 3 weeks before breaking up completely. Although entire spongioblast colonies undergo changes, only an occasional astrocyte exhibits CPE. Individual cells become greatly en-
658
PADGETT, ROGERS,
AND WALKER
INFECT. IMMUN.
FIG. 1. PHFG cells in culture. (A) Uninoculated, control culture containing a dense colony of spongioblasts; (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 and deeply staining, bizarre-shaped, or multinucleated. In maintenance medium the altered astrocytes neither multiply nor become necrotic during 4 to 5 weeks of incubation. Virion (V) antigens, detected by immunofluorescent staining, are found in both types of cells (Fig. 2), and antigen is detected in many astrocytes that do not show enlargement. However, not all cells in the PHFG cell cultures appear to be susceptible to JCV because only 75 to 90% of them contain V antigen 3 weeks after inoculation. Progeny virus, as measured by HA, is associated strongly with the infected cells and cell debris. Only low levels of HA are ever detected in the culture fluid and then only when much debris is present. JCV does not appear to spread readily from cell to cell except for those within a spongioblast colony. If a low-titered inoculum (10 HAU/250-ml flask) is added to cultures containing isolated spongioblast colonies, only some of the colonies will exhibit CPE 5 weeks later. Also, in establishing the fluorescent-cell assay of infectivity, no evidence of secondary infection in the form of clusters of antigen-containing cells was seen until 10 or 11 days postinoculation. Good yields of HA, 200fold over input, are obtained only from PHFG cell cultures that are composed predominately of spongioblasts. To obtain the highest HA yields, it is necessary to process the infected cultures as described in the preparation of JCV pools (9, 11).
FIG. 2. JCV virion (V) antigen in nuclei of PHFG cells 14 days after inoculation demonstrated by immunofluorescence. x320.
Time of appearance of T and V antigens. Because JCV appeared to grow slowly in PHFG cell cultures, the time of appearance of T and V antigens after infection of cells was determined. For this experiment, replicate PHFG cell cultures were grown on 12-mm cover slips and inoculated with 4 infectious units of JCV/ cell. After an adsorption period, a potent JCV-
CHARACTERISTICS OF JC VIRUS
VOL. 15, 1977
neutralizing antiserum was added to some of the cultures. At 24-h intervals for 5 days four cultures were fixed in acetone for 10 min. Two cover slips were stained for T antigen and two were stained for V antigen. The percentages of cells containing T and V antigens were determined by examining the cultures at a magnification of x320. At 24 h postinoculation less than 1% of PHFG cells contained detectable levels of T antigen (Table 1). The percentage of T antigen-containing cells increased daily for 5 days even in the presence of neutralizing antibody. Many astrocytes contained T antigen; in fact, the first positive cells were astrocytes. V antigen appeared later than T antigen. A few cells had produced V antigen at 48 h postinoculation, but even at 5 days postinoculation, when close to 60% of the cells contained T antigen, only approximately 2% contained V antigen. Although the multiplicity of infection used was probably not great enough to infect all of the cells, any cell producing V antigen would be detected because every cell was examined. Hemagglutinin. Pools ofJCV exhibit hemagglutinating activity that has been shown to be a property of the infectious virion (9). JCV was found to agglutinate human, guinea pig, and chicken erythrocytes, but not those of hamsters, sheep, African green monkeys, or rhesus monkeys. The erythrocytes are usable up to 7 days after collection. The hemagglutinin was not inactivated by heating at 56° C for 7 h, but it was destroyed when held at 80'C for 15 min. Host cell range of JCV. Our attempts to TABLE 1. Time of appearance of T and V antigens in JCV-infected PHFG cells Percentage of cells containing
antigen
Days after inoculation T antigena 1
V
antigen
1 0 0.05 2 10 36 0.22 3 30 0.17 3 + antiserum' 4 46 1.0 57 2.3 5 NCd 5 + antiserum 59 a Percentage of T antigen-containing cells is based on count of antigen-containing cells/100 cells in each of five widely separated areas of the culture. b The entire culture was examined and the V antigen-containing cells were counted. Percentages are that number per average number of cells per culture (3 x 104). e Medium containing antiserum sufficient to neutralize 106 fluorescent-cell units of JCV was added after adsorption on day zero and again on days 2 and
4. d
Not counted.
659
cultivate JCV in cells other than PHFG cells have failed. No CPE has been observed in unstained cultures of the following cells after inoculation with 100 HAU of JCV: primary human embryonic kidney, lung, intestine, liver, and amnion; primary human adult testes; two human diploid cells (L-809 and WI-38); a human heteroploid cell (Chang conjunctiva); primary African green monkey kidney cells; BSC-1, CV1, and Vero cells; adult rhesus monkey glial cells; hamster fetal glial cells; adult mink glial cells; mouse embryo cells. All of the inoculated cell cultures were held for a minimum of 21 days at 370C. At the end of the observation period all cultures were tested for HA activity and all were negative. For the following cells a serial blind passage was made: primary human embryonic kidney, lung, intestine, liver, and amnion; WI-38 and Chang conjunctiva; primary African green monkey kidney, CV-1, and Vero; hamster fetal glial cells. These cultures were also observed for 21 days, and again no CPE or HA was detected. In other experiments, cultures of WI-38, Vero, CV-1, and primary cultures of human embryonic kidney, lung, intestine, and liver cells were each inoculated with 100 HAU of JCV and stained for JCV T and V antigens 10 and 21 days later. At 10 days postinoculation a few cells 'containing T and a few containing V antigen were observed in all of the cultures of human cells, except primary human embryonic lung cells, but no antigencontaining cells were seen in the Vero or CV-1 cultures. The number of cells containing either antigen did not increase between 10 and 21 days. Stability of JCV. The sensitivity of JCV to ethyl ether was determined by comparing the infectivity titer of a sample of virus after exposure to ether with that of a sample not so treated. Mixtures of equal volumes of virus suspension and either anesthetic ether or phosphate-buffered saline (PBS) were held at 40C for 18 h with continuous agitation. After exposure to ether, the sample contained 104-5 TCID50/ml as compared with 104 3 TCID5dml in the 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 sonically treated briefly. Samples were diluted with equal volumes of PBS or phosphate-buffered 2 M MgCl2 and heated at 500C for 1 h. The TCID50 of the virus suspension both before and after heating in PBS was 106/ml, but after heating in 1 M MgCl2 it was less than 102/ml. Heat lability in the presence of MgCl2 is a characteristic of polyomaviruses (7). Serological relationships with other papovaviruses. Initially, using immunofluorescent
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PADGETT, ROGERS, AND WALKER
techniques, we found no serological relationship between the V antigen(s) of JCV and other papovaviruses including the simian polyomavirus SV40 (11). Later, it was reported that crossreactions did occur between JCV and both SV40 and BKV, another human polyomavirus, when hyperimmune sera were used in immune agglutination tests (1, 13). Therefore, we reexamined the serological relationship of JCV to other papovaviruses and to SV40 and BKV in particular, using antisera against V antigen(s) of various papovaviruses. Since SV40 does not hemagglutinate, it was not possible to use the simple and quick HI titration with all three viruses. The HI test was used with BKV, neutralization with SV40, and both tests were done with JCV so that the titers obtained with JCV could be compared directly with those obtained with the other two viruses. From the results (Table 2) it is obvious that JCV is a distinct virus but one that does have a minor relationship to both SV40 and BKV. There was great variation among the antisera in the extent to which they reacted with the heterologous viruses; however, within each group of JCV, BKV, and SV40 antisera, one or more individual antiserum(a) did react with the heterologous viruses. Specifically, all of the BKV and
INFECT. IMMUN.
SV40 antisera tested neutralized JCV to some extent although not all of them prevented HA by JCV. No reactions were observed between JCV and antisera against polyoma virus, K virus, or human papilloma virus. Is JCV a "new virus"? Because of the weak antigenic relationship between JCV and the simian polyomavirus SV40, the possibility that JCV evolved from SV40 after the introduction of the latter virus into large numbers of humans via contaminated lots of poliovirus vaccines was tested. Sera obtained locally before the introduction of poliovirus vaccines were titrated for HI antibodies against JCV. Forty-six of 50 sera (92%) obtained from young adults in 1950 and stored frozen had HI titers of 64 or greater against JCV. Thus, infection of humans with JCV was common before the introduction of poliovirus vaccines in 1954. Search for HI antibodies in sera from primates and other animals. Serological evidence (10) indicates that infection with JCV is common during childhood, but little else is known concerning the epidemiology of the infection. The possibility of an animal reservoir for JCV was investigated by testing samples of sera from a variety of animals for HI antibodies against JCV. Sera tested included samples
TABLE 2. Serological relationship of JCV to SV40, BKV, and other papovaviruses Test virus
Serum against:
JCV
SV40
BKV
HI tite' HI titer 5.1 x 105 2.0 x 106 512 3.4 x 105 6.4 x 104
