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A polyoma virus variant with new antigenic determinants
VIROLOGY
19
(1962)
DISCUSSION AND PRELIMINARY A Polyoma
Virus Variant
Antigenic
with
New
Determinants’
Recent studies have described two polyoma virus (PV) mutants and outlined several of their biological characteristics (IS). Briefly, they differ in that one forms a large plaque in mouse embryo cells, produces high titers of hemagglutinins (HA), and is highly oncogenic for newborn mice and hamsters. The second forms small plaques, produces lower titers of HA, and is of a lower oncogenic activity. No antigenie difference between the two mutants has been shown. Furthermore, the largeplaque mutant is entirely replaced by the small-plaque virus in situations in which mouse and hamster cells become persistently infected with PV (1, 3). We have encountered a similar situation in which persistently infected L cells yielded a variant of PV which has several characteristics in common with the small-plaque virus above, but which has a distinct antigenie specificity demonstrable by hemagglutination-inhibition (HI) tests and confirmed by neutralization and absorption tests. The variant was selected for by growth in L cell cultures (strain 929 of Earle) which had been inoculated with PV strain 210 received from Dr. Bernice Eddy and passed 3 times in mouse embryo cell cultures. It was first recognized by its failure to produce hemagglutinins (HA) in P388D1 cells, although it produced cytopathic effects and was capable of proliferating to titers of lo6 and IO7 TCDBO per milliliter. Since it produced HA in L cells, the antigenic structure could be studied using hemagglutination-inhibition (HI) 1 Supported by Research Grant C-2223 and C5206, and Special Research Fellowship SG 13,959 from the National Institutes of Health, United States Public Health Service, and the Nelson B. Delavan Virus Research Fund.
REPORTS
tests, which will be described. The time period required to select the variant is not known, although within the first 60 days, during which time the carrier cell culture was subcultured once, the H+ to H- variation had occurred (see below). Table 1 illustrates the results of HI tests performed simultaneously in which three different sera were tested for HI antibody using 8 HA units of 3 different antigens, i.e., SE-3049-H+ and SE-210-H+ which are two strains of PV received from Dr. Bernice Eddy and SE-210-H-, the variant derived from SE-210-H+ after propagation in L cells. It will be noted that the patterns of antibody directed against each antigen were distinctly different in the three sera. The guinea pig antiserum to Dulbecco’s largeplaque mutant reacted maximally with the SE-3049-H+ and SE-210-H+ antigens and repeatedly reacted to a four- to eightfold lower titer with the variant SE-210-H-. Antisera to SE-210-H+, the parent stock from which the variant was derived, did not distinguish between the three antigens, and antisera to the SE-%lO-H- variant itself showed a high homotypic reaction with the lowest cross reaction to the SE-3049-H+ virus. Neutralization tests in P388Di cell tubes performed on several of these sera reflect a similar relationship (Table 2). Absorption studies have been carried out in which rabbit antiserum to the variant, SE-210-H-, was absorbed for 24 hours with tissue culture fluids containing high HA titers of either SE-3049-H+ or the variant and the virus particle-antibody complexes were removed by centrifugation at 97,000 g for 3 hours. As a control, antiserum was absorbed with uninfected tissue culture fluids and treated in the same fashion. As seen in Table 1, absorption of this serum with heterologous antigen, SE-3049-H+, reduced the titer to this antigen more than 94%, while reducing that to the variant 105
106
DISCUSSION
AND
PRELIMINARY
REPORTS
TABLE 1 TITERS OF ANTISERA
HEMAGGLUTINATI~N-INHIBITION
TO POLYONA VARIANTS
HI Units?/.25
HA Antigens (8HA) Anti-PV1pb SE-3049.Hfd
ml Anti-SE-210.H+c
25,600 SD = f0.54 tubes 25,600 F,400 SD = ~0.54 tubes
SE-210-H+d SE-210-H-a
HI Titer’ of Anti-SE-210-H-
Anti-SE-210-H-c
1600
400
3200 3200
800 3200
rabbit serum absorbed with
SE-3049-H+
SE-210-H-
HA Antigen @HA)
SE-210-HSE-210-H+ SE-3049-H+
Control
Absorbed
Fold reduction
“/b Reduction
2,560 640 320
640 80 20
4 8 16
75 87 94
Control
2,560 640 320
Absorbed
80 80 40
Fold reduction
% Reduction
32 8 8
97 87 87
a Expressed as reciprocal of two-fold serum dilutions. b Guinea pig antisera to Dulbecco’s large-plaque mutant propagated in hamster embryo cell culture, kindly provided by Dr. Matilda Benyesh-Melnick. c Rabbit antisera to the antigens indicated, SE-210-H+ propagated in mouse embryo cells, SE-210-Hpropagated in L cells and extracted once with genitron. Animals given a series of intravenous and intraperitoneal injections over 2-month period. d Polyoma virus strains provided by Dr. Bernice Eddy and propagated in P388D1 cells. 6 Polyoma virus propagated in an L cell carrier culture-59th passage. TABLE
2
ANTIBODY TITERS OF ANTISERA TO POLYOMA VARIANTS~ Neutralization (Challenge virus 100-1000 TCDSO)
Anti-PV’P I
SE-3049-H+ SE-210-H-
titer per 0.4 ml
I 3200 400
Anti-SE210-H800 6400
HI Units per 0.25 ml HA Antigens (8 HA)
SE-210-HPV ip-Dulbeceo* PVaP-Dulbecco*
Anti-SE210-H12,aOO 51,200 25,600
1600 4OQ 400
(1See Table 1 for explanatory footnotes. * Polyoma mutant strains, large plaque (lp) and small plaque (sp) received from Dr. Dulbecco, propagated in mouse embryo cells.
75%. On the contrary, absorption with the homotypic antigen resulted in a 97% reduction in homotypic antibody and an 87% reduction in titer to the other antigen. Two other genetic characters of the SE210-H- variant are of interest. The first pertains to its reduced oncogenic capacity for hamsters less than 7 days old. This agent produced intraperitoneal tumors in less than 50% of such animals in 2 to 3 months with only an occasional hemorrhagic death from a cystic liver lesion when 6.5 to 7.5 log10 TCl&, were injected intraperitoneally. In contrast, the H+ strains, either SE-3049-H+ or SE-210-H+, produced disseminated tumors in 100% of animals which survived an early demise from the hemorrhagic lesion when doses such as 6.0 to 6.5 log10 TCDBO were injected. The second interesting character of SE-210-Hpertains to its inability to produce measurable HA in P388D1 or mouse embryo cells (which has been chosen as the criterion for the H-
DISCUSSION
AND
PRELIMINARY
character), in spite of the fact that its replication in L cells is accompanied by HA production. We have not yet satisfactorily ascertained the plaque size character of the variant SE-210-H-. The antigenic and HA characters of PV mutants that might be selected from virus populations on passage in L cells became of interest since it had been shown that smallplaque variants usually become predominant whenever persistent infection is established in mouse cells (1, 3). Would conditions which allow selection of small plaque particles also select populations with the altered antigenic structure as well as the change from H+ to H-? It was found that SE-3049-H+ virus could be carried for 18 L cell subcultures (110 days) without becoming H- and for 12 subcultures without reacting antigenically as the SESlO-Hvariant. It would appear then that prolonged passage in L cells need not select out variants with different characters such as H- and the new antigenic specificity. We have tested the antigenic reactivity of 2 plaque-purified strains of PV derived by Dulbecco and supplied through the kindness of Drs. Wenzel and Dulbecco. The first, a large-plaque mutant, designated here PV’p, is H+ and highly oncogenic for hamsters in our hands. The second, a smallplaque mutant, designated PV*p, is more variable in its “H” characteristic, but does produce HA in mouse embryo and P388D1 cells. It is also quite oncogenic. As shown in Table 2, both variants behave in general as the SE-3049-H+ strain except for the intermediate position of PVs*’ when tested against the guinea pig antiserum to the large plaque agent itself. In summary, a variant of polyoma virus has been described, SE-210-H-, which differs from the strain from which it was derived, SE-210-H+, by the presence of an apparently new antigenic determinant as well as by the inability to produce HA in P388D1 or mouse embryo cells (H-) and a reduced oncogenic capacity for newborn hamsters. REFERENCES 1. MEDINA, D., and SACHS, L., Virology (1960).
10, 387-388
107
REPORTS
2. GOTLIEB-STEMATSKY, T., and LEVENTON, S., %t. J. Exptl. Pathol. 41,507-519 (1960). 3. DULBECCO, R., and VODT, M., Proc. Natl. Acad. Sei. U. 8. 46, 1617-1623 (1960). J. DONALD HARE HERBERT R. MORGAN Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester, New York Accepted October 19, 1962
Transformation
in Hamster
by Vacuolating
Kidney Virus,
Monolayers
W-40
Transformation, or the changes induced in morphology and growth characteristics of cells grown in tissue culture by the action of viruses, has been reported with various tumor viruses (1-B). Recently, it has been demonstrated that simian virus-40 (vacuolating virus) (9), which is tumorigenic in hamsters in viva (10, 11), has the ability to cause transformation in explants of human tissue such as the buccal mucosa, skin (12)) and human embryonic kidney cultures (13-16). This preliminary communication describes transformation in hamster kidney monolayer cultures by SV-40. Strain 777 of SV-40 was obtained from Dr. P. Gerber and was passed three times in African green (Cercopithecus) monkey kidney cultures. This pool was tested extensively for indigenous simian agents and none were detected. Primary hamster (Mesocricetus auratus) kidney monolayers, maintained on Eagle’s Basal Medium with nonessential amino acids, 5% horse serum, penicillin (100 U/ml), and streptomycin (100 pg/ml) , were inoculated with 10B.j TCIDS,, of SV-40. A 24-hour period of time for adsorption was allowed. The supernatant was then discarded and the cell sheet was washed with BME and horse serum 5%. Uninoculated controls were similarly treated. Fluids were changed twice weekly. By the 18th-20th day a small number of multinucleated cells 2-4 times the size of normal cells were seen in the inoculated tubes. During the first 30 days after inoculation there was slow “nonspecific” degen-
19
(1962)
DISCUSSION AND PRELIMINARY A Polyoma
Virus Variant
Antigenic
with
New
Determinants’
Recent studies have described two polyoma virus (PV) mutants and outlined several of their biological characteristics (IS). Briefly, they differ in that one forms a large plaque in mouse embryo cells, produces high titers of hemagglutinins (HA), and is highly oncogenic for newborn mice and hamsters. The second forms small plaques, produces lower titers of HA, and is of a lower oncogenic activity. No antigenie difference between the two mutants has been shown. Furthermore, the largeplaque mutant is entirely replaced by the small-plaque virus in situations in which mouse and hamster cells become persistently infected with PV (1, 3). We have encountered a similar situation in which persistently infected L cells yielded a variant of PV which has several characteristics in common with the small-plaque virus above, but which has a distinct antigenie specificity demonstrable by hemagglutination-inhibition (HI) tests and confirmed by neutralization and absorption tests. The variant was selected for by growth in L cell cultures (strain 929 of Earle) which had been inoculated with PV strain 210 received from Dr. Bernice Eddy and passed 3 times in mouse embryo cell cultures. It was first recognized by its failure to produce hemagglutinins (HA) in P388D1 cells, although it produced cytopathic effects and was capable of proliferating to titers of lo6 and IO7 TCDBO per milliliter. Since it produced HA in L cells, the antigenic structure could be studied using hemagglutination-inhibition (HI) 1 Supported by Research Grant C-2223 and C5206, and Special Research Fellowship SG 13,959 from the National Institutes of Health, United States Public Health Service, and the Nelson B. Delavan Virus Research Fund.
REPORTS
tests, which will be described. The time period required to select the variant is not known, although within the first 60 days, during which time the carrier cell culture was subcultured once, the H+ to H- variation had occurred (see below). Table 1 illustrates the results of HI tests performed simultaneously in which three different sera were tested for HI antibody using 8 HA units of 3 different antigens, i.e., SE-3049-H+ and SE-210-H+ which are two strains of PV received from Dr. Bernice Eddy and SE-210-H-, the variant derived from SE-210-H+ after propagation in L cells. It will be noted that the patterns of antibody directed against each antigen were distinctly different in the three sera. The guinea pig antiserum to Dulbecco’s largeplaque mutant reacted maximally with the SE-3049-H+ and SE-210-H+ antigens and repeatedly reacted to a four- to eightfold lower titer with the variant SE-210-H-. Antisera to SE-210-H+, the parent stock from which the variant was derived, did not distinguish between the three antigens, and antisera to the SE-%lO-H- variant itself showed a high homotypic reaction with the lowest cross reaction to the SE-3049-H+ virus. Neutralization tests in P388Di cell tubes performed on several of these sera reflect a similar relationship (Table 2). Absorption studies have been carried out in which rabbit antiserum to the variant, SE-210-H-, was absorbed for 24 hours with tissue culture fluids containing high HA titers of either SE-3049-H+ or the variant and the virus particle-antibody complexes were removed by centrifugation at 97,000 g for 3 hours. As a control, antiserum was absorbed with uninfected tissue culture fluids and treated in the same fashion. As seen in Table 1, absorption of this serum with heterologous antigen, SE-3049-H+, reduced the titer to this antigen more than 94%, while reducing that to the variant 105
106
DISCUSSION
AND
PRELIMINARY
REPORTS
TABLE 1 TITERS OF ANTISERA
HEMAGGLUTINATI~N-INHIBITION
TO POLYONA VARIANTS
HI Units?/.25
HA Antigens (8HA) Anti-PV1pb SE-3049.Hfd
ml Anti-SE-210.H+c
25,600 SD = f0.54 tubes 25,600 F,400 SD = ~0.54 tubes
SE-210-H+d SE-210-H-a
HI Titer’ of Anti-SE-210-H-
Anti-SE-210-H-c
1600
400
3200 3200
800 3200
rabbit serum absorbed with
SE-3049-H+
SE-210-H-
HA Antigen @HA)
SE-210-HSE-210-H+ SE-3049-H+
Control
Absorbed
Fold reduction
“/b Reduction
2,560 640 320
640 80 20
4 8 16
75 87 94
Control
2,560 640 320
Absorbed
80 80 40
Fold reduction
% Reduction
32 8 8
97 87 87
a Expressed as reciprocal of two-fold serum dilutions. b Guinea pig antisera to Dulbecco’s large-plaque mutant propagated in hamster embryo cell culture, kindly provided by Dr. Matilda Benyesh-Melnick. c Rabbit antisera to the antigens indicated, SE-210-H+ propagated in mouse embryo cells, SE-210-Hpropagated in L cells and extracted once with genitron. Animals given a series of intravenous and intraperitoneal injections over 2-month period. d Polyoma virus strains provided by Dr. Bernice Eddy and propagated in P388D1 cells. 6 Polyoma virus propagated in an L cell carrier culture-59th passage. TABLE
2
ANTIBODY TITERS OF ANTISERA TO POLYOMA VARIANTS~ Neutralization (Challenge virus 100-1000 TCDSO)
Anti-PV’P I
SE-3049-H+ SE-210-H-
titer per 0.4 ml
I 3200 400
Anti-SE210-H800 6400
HI Units per 0.25 ml HA Antigens (8 HA)
SE-210-HPV ip-Dulbeceo* PVaP-Dulbecco*
Anti-SE210-H12,aOO 51,200 25,600
1600 4OQ 400
(1See Table 1 for explanatory footnotes. * Polyoma mutant strains, large plaque (lp) and small plaque (sp) received from Dr. Dulbecco, propagated in mouse embryo cells.
75%. On the contrary, absorption with the homotypic antigen resulted in a 97% reduction in homotypic antibody and an 87% reduction in titer to the other antigen. Two other genetic characters of the SE210-H- variant are of interest. The first pertains to its reduced oncogenic capacity for hamsters less than 7 days old. This agent produced intraperitoneal tumors in less than 50% of such animals in 2 to 3 months with only an occasional hemorrhagic death from a cystic liver lesion when 6.5 to 7.5 log10 TCl&, were injected intraperitoneally. In contrast, the H+ strains, either SE-3049-H+ or SE-210-H+, produced disseminated tumors in 100% of animals which survived an early demise from the hemorrhagic lesion when doses such as 6.0 to 6.5 log10 TCDBO were injected. The second interesting character of SE-210-Hpertains to its inability to produce measurable HA in P388D1 or mouse embryo cells (which has been chosen as the criterion for the H-
DISCUSSION
AND
PRELIMINARY
character), in spite of the fact that its replication in L cells is accompanied by HA production. We have not yet satisfactorily ascertained the plaque size character of the variant SE-210-H-. The antigenic and HA characters of PV mutants that might be selected from virus populations on passage in L cells became of interest since it had been shown that smallplaque variants usually become predominant whenever persistent infection is established in mouse cells (1, 3). Would conditions which allow selection of small plaque particles also select populations with the altered antigenic structure as well as the change from H+ to H-? It was found that SE-3049-H+ virus could be carried for 18 L cell subcultures (110 days) without becoming H- and for 12 subcultures without reacting antigenically as the SESlO-Hvariant. It would appear then that prolonged passage in L cells need not select out variants with different characters such as H- and the new antigenic specificity. We have tested the antigenic reactivity of 2 plaque-purified strains of PV derived by Dulbecco and supplied through the kindness of Drs. Wenzel and Dulbecco. The first, a large-plaque mutant, designated here PV’p, is H+ and highly oncogenic for hamsters in our hands. The second, a smallplaque mutant, designated PV*p, is more variable in its “H” characteristic, but does produce HA in mouse embryo and P388D1 cells. It is also quite oncogenic. As shown in Table 2, both variants behave in general as the SE-3049-H+ strain except for the intermediate position of PVs*’ when tested against the guinea pig antiserum to the large plaque agent itself. In summary, a variant of polyoma virus has been described, SE-210-H-, which differs from the strain from which it was derived, SE-210-H+, by the presence of an apparently new antigenic determinant as well as by the inability to produce HA in P388D1 or mouse embryo cells (H-) and a reduced oncogenic capacity for newborn hamsters. REFERENCES 1. MEDINA, D., and SACHS, L., Virology (1960).
10, 387-388
107
REPORTS
2. GOTLIEB-STEMATSKY, T., and LEVENTON, S., %t. J. Exptl. Pathol. 41,507-519 (1960). 3. DULBECCO, R., and VODT, M., Proc. Natl. Acad. Sei. U. 8. 46, 1617-1623 (1960). J. DONALD HARE HERBERT R. MORGAN Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester, New York Accepted October 19, 1962
Transformation
in Hamster
by Vacuolating
Kidney Virus,
Monolayers
W-40
Transformation, or the changes induced in morphology and growth characteristics of cells grown in tissue culture by the action of viruses, has been reported with various tumor viruses (1-B). Recently, it has been demonstrated that simian virus-40 (vacuolating virus) (9), which is tumorigenic in hamsters in viva (10, 11), has the ability to cause transformation in explants of human tissue such as the buccal mucosa, skin (12)) and human embryonic kidney cultures (13-16). This preliminary communication describes transformation in hamster kidney monolayer cultures by SV-40. Strain 777 of SV-40 was obtained from Dr. P. Gerber and was passed three times in African green (Cercopithecus) monkey kidney cultures. This pool was tested extensively for indigenous simian agents and none were detected. Primary hamster (Mesocricetus auratus) kidney monolayers, maintained on Eagle’s Basal Medium with nonessential amino acids, 5% horse serum, penicillin (100 U/ml), and streptomycin (100 pg/ml) , were inoculated with 10B.j TCIDS,, of SV-40. A 24-hour period of time for adsorption was allowed. The supernatant was then discarded and the cell sheet was washed with BME and horse serum 5%. Uninoculated controls were similarly treated. Fluids were changed twice weekly. By the 18th-20th day a small number of multinucleated cells 2-4 times the size of normal cells were seen in the inoculated tubes. During the first 30 days after inoculation there was slow “nonspecific” degen-