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Defective interfering particles from poliovirus vaccine and vaccine reference strains
VIROLOGY
60,579-583
(1974)
Defective
Interfering
Particles
and Vaccine LEROY
Reference
C. McLAREN
Department of Biology, Uniuersity of California Microbiology, I/niuersit.y of New Mexico
From Poliovirus
AND
JOHN
Strains J. HOLLAND
at San Diego, La Jolla, California School of Medicine, Albuquerque.
Accepted
April
Vaccine
92097 and Department New Mexico 87231
of
25. 1974
Defective interfering (DI) viral particles have been found to be associated with attenuated oral poliovirus vaccine and also with poliovirus vaccine reference strains. The DI particles replicate in low-passage human fibroblastic cells, the RSC-1 line of monkey kidney cells as well as in HeLa cells. DI particles of the attenuated polioviruses are generated after relatively few serial, high multiplicity passages of cloned, DI-free vaccine virus, whereas virulent strains of poliovirus generate a significant number of DI only after a large number of passages.
Defective interfering (DI) viral particles have been described for many classes of animal viruses (I, 2) and it has been suggested that they may play a significant role in viral diseases in uiuo (2). In support of this suggestion, it has recently been shown that defective T particles of vesicular stomatitis virus (VSV) provide protection of mice against viral disease and death following intracerebral inoculation of standard, infectious VSV (3). DI particles have been demonstrated by Cole and Baltimore using stocks of virulent type 1 poliovirus (Mahoney strain) (4). These DI particles were found to be generated after approximately 20 passages of virus at high multiplicity of infection of HeLa cells. They appear to be deletion mutants which lack about 15% of the RNA of standard virus and their density at equilibrium is lower in CsCl gradients. The DI particles contain the same capsid proteins as standard poliovirus, and they are efficiently adsorbed to HeLa cells and become uncoated. Being defective, they require coinfection of cells by standard virus to provide a helper-function for DI particle formation. In cells infected only by poliovirus DI particles, the capsid protein precursor (NCVP-l), capsid proteins and
therefore intact virions are absent, although other viral proteins encoded by the defective RNA genome are demonstrable (5, 6). In the present report, we show that DI particles are present in commercial preparations of oral poliovirus vaccine and also in poliovirus vaccine reference standards. The DI particles in attenuated poliovirus strains replicate in monkey kidney and human fibroblast cells as well as in HeLa cells. They appear to be generated more quickly in cells infected with attenuated poliovirus strains than with virulent type 1 poliovirus. BSC-1, and NHP (primary human skin fibroblasts, passage 7) cells were grown as monolayers in screw-cap glass bottles in Eagle’s medium containing 5% calf serum (MEM). Virulent poliovirus, type 1, was a clinical isolate previously typed by Dr. Edwin Lennette. Attenuated, vaccinereference strains of poliovirus, type 1 (TA3), type 2 (TB3) and type 3 (TC3) were kindly supplied by the Division of Biological Standards of the U.S.F.D.A. Trivalent, oral poliovaccine was from a commercial lot prepared by Wyeth Laboratories. For uridine labeling of viruses, approximately 2 x lo6 cells were infected at a multiplicity
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of 100, and after adsorption at room temperature for 30 min, Eagle’s minimalessential-medium containing 2%) dialyzed calf serum, 5 pg of actinomycin D per ml and 200 PCi of [5-3H Juridine per ml was added. Following incubation at 37” for 4 hr radioactive media was removed, cells were rinsed with 0.15 M NaCl and then reincubated for 12 hr in MEM. Virus plaque assays were performed on HeLa cell monolayers under 0.4?~ agarose (7). Labeled polioviruses were purified by a modification of the method of Hall and Rueckert (8). Infected cells were disrupted by freezing and thawing five times and were then dialyzed overnight against 0.15 M NaCl containing lo- 3 M EDTA. Cell debris was then removed by centrifugation at 2000 R for 10 min. Nonlabeled, virulent, poliovirus, 10” PFU, was added as carrier, and the viruses were concentrated by centrifugation at 146,000 g for 90 min. Virus pellets were then suspended in 0.15 M NaCI, 0.2 M sodium phosphate pH 7.2 (PBS) and treated with ribonuclease, 10 pg per ml for 30 min at 25’ and further purified by gel filtration through Sepharose 2B (Pharmacia) columns equilibrated with PBS. Eluted viruses were then banded in CsCl (p = 1.33-1.34) by centrifugation at 140,000 R in a type 50 Ti rotor for 16 hr at 4”. Gradients were collected by puncturing the tubes at the bottom. Each fraction was precipitated by the addition of 10%~trichloracetic acid (TCA) and carrier (bovine serum albumin and yeast RNA, 25 pg of each per ml). Radioactivity in each TCA insoluble fraction collected was determined by dissolving the TCA precipitates in NCS tissue solubilizer (Amersham/ Searle) followed by the addition of Omnifluor (New England Nuclear). To determine the possible presence of DI particles in oral poliovaccine, we inoculated undiluted trivalent oral poliovaccine (0.2 ml) onto approximately lo6 HeLa cells. After 2 hr adsorption at 37” fresh medium was added and the culture incubated at 37”. After apparent cytopathic destruction of the cell monolayer undiluted cell culture fluid was reinoculated onto new HeLa monolayers and [3H]uridine labeled. The labeled, purified viral progeny was
found to contain a peak of radioactivity of similar density of that found for DI particles (Fig. 1). The densities of the virion and DI particle peaks were found to be 1.34 and 1.31, respectively, which is consistent with those reported for virulent type 1 poliovirus and its DI (4-6). Neutralization of more than 90% of the virus in the second HeLa cell passage of the vaccine virus was observed when reference type 1 poliovirus antiserum was employed in a plaque reduction assay. We next examined each of the three reference strains used fbr vaccine production. Reference strains, TA3, TB3 and TC3 of poliovirus types 1, 2 and 3 respectively, during second passage in HeLa cells, were labeled with [3H]uridine as described. Analysis of CsCl gradients for radioactivity revealed that each of the three reference viruses also generated DI particles as shown in Fig. 2. It will be noted that the second passage of TB2 in HeLa cells differs from TA3 and TC3 strains in that there was no significant amount of radioactivity in the virion peak. Infectivity assays on fractions collected from the p = 1.33-1.34 region of the gradient demonstrated little or no infectious virus was synthesized. This result was found not to be limited to strain TB3 and was also found occasionally with TA3 and TC3 in other experiments. This effect is
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FIG. 1. Appearance ot’D1 particles associated with attenuated. oral poliovaccine. Second HeLa cell passage of vaccine virus, labeled with [“Hluridine was centrifuged at 146,000 p for 16 hr at 4’ in CsCi. p 1.93-l 34, (Beckman rotor Ti 501, collected into 0.3 ml fractions and analyzed for radioactivity. Higher density is at the left.
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FIG. 2. DI particles associated with poliovirus reference strains. A attenuated strain X3. Second HeLa cell passage of each virus was labeled with [3H]uridine Fig. 1.
likely related to the large ratio of DI to infectious virus particles in the inoculum resulting in interference as reported for virulent poliovirus (6). Analysis of virulent, type 1 poliovirus infected cells, included as a control with each experiment, consistently yielded a prominent virion and only a minor DI particle profile (Fig. 3). Because DI particles have thus far been described only for poliovirus replicating in HeLa cells, reference strains TA3, TB3 and TC3 of monkey kidney (MoK) cell origin were [3H]uridine labeled during second passage in either the BSC-1 line of MoK cells or in the low passage, fibroblasts. line NHP. DI particles were found to be produced by all three virus strains following infection of both BSC-1 and NHP cells. Fig. 4 illustrates the appearance of both virion and DI particle peaks in BSC-1 and NHP cells infected by TA3 virus and is similar to those observed previously for virus propagated in heteroploid HeLa cells. In addition, it was consistently observed that most of the radiolabel was present in the DI particle peak of virus-infected NHP cells. I n previous studies (4-6) it has been
*-
strain TA3; B strain TB8; C and analyzed as described in
t
CT ‘0 I
FE. 3. C&l density-gradient centrifugation ol virulent type 1 poliovirus, (3H]uridine labeled after 35 passages in HeLa cells. Gradient was analyzed as described in Fig. 1.
reported that a virulent strain of plaque purified type 1 poliovirus required 20~25 passages in HeLa cells before significant amounts of DI particle were apparent. Using our virulent strain of type 1 poliovirus only small quantities of labeled DI particles are generated in HeLa cells even after 90 serial passages. When we plaque-purified each of the poliovaccine reference strains the DI peaks were eliminated during the first few serial undiluted passages (see Fig. 5A). However,
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t
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i
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fection of HeLa, BSC-1 or human diploid fibroblast cells. These DI showed the typical DI behavior of disappearing after cloning, and reappearing upon further serial high multiplicity passage. Using these plaque-purified poliovirus reference strains, it was clear that fewer serial passages were required for the appearance of DI particles than has been observed for virulent strains of poliovirus. It is conceivable that the relatively limited replication in vim of attenuated vaccine strains of poliovirus may be related to their apparent genetic potential for more rapid generation of DI particles. It is interesting that the diploid human fibroblasts generated a larger proportion of DI particles than did the HeLa or BSC-1 lines infected with the same virus pool (see Fig. 4). A similar influence of host cell type upon autointerference has been observed in other virus groups generating DI particles (2, 9. IO). There is presently no evidence regarding whether DI particles play any role in the ,7
FIG. 4. Production of DI particles of TA3 poliovirus in other cell lines. Both A: (HSC-1 cells) and B (NHP cell line) were [3H]uridine in the second passage of the reference virus cell type.
strain of infected labeled in each
using this plaque-purified virus, DI particles were found to be present after as few as five serial passages in HeLa cells. After a single passage in NHP cells of the HeLa passage 5, plaque-purified TA3 virus, most of the radiolabel was found to be in the DI particle peak as shown in Fig. 5B. The generation of DI particles by virulent strains of type 1 poliovirus has been shown to require many successive passages of virus at high multiplicity (4). It is therefore not surprising that such particles might be present in attenuated strains of poliovirus which have undergone many passages in cell culture prior to their use in vaccine preparation. Our analysis of commercially distributed oral poliovaccine prepared in monkey kidney cell cultures, as well as reference strains of types 1, 2 and 3 poliovirus shows that significant amounts of DI particles are demonstrable after in-
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FIG. 5. Dl particles associated with plaque purified TA3 strain poliovirus. (A) Plaque purified virus radiolabeled after infection of NHP cells, following one passage in HeLa cells; (B) plaque purified virus after five serial passages in HeLa cells and a single passage in NHP cells. Infected cells were [3H]uridine labeled and centrifuged in CsCl as previously described.
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attenuation of vaccine strains, or in the production of immunity following ingestion of attenuated, oral poliovaccine. It is noteworthy that defective T particles protect mice prophylactically against lethal infection following intracerebral inoculation of VSV. Mice inoculated with virus-free, T particles of WV have also been shown to develop immunity to challenge 10 days later with homologous virus (3). It would be informative to determine whether poliovirus vaccine strains are more virulent in the intraspinal monkey test after they are freed of DI particles by cloning, and, if so, whether addition of purified DI particles to the cloned virus restores attenuation. ACKNOWLEDGMENTS We thank Estelle Bussey for excellent technical assistance, and Dr. Michael Doyle for helpful discus
sions. Supported by Grant tional Cancer Institute.
CA 10802 from the Na-
REFERENCES 23. 293 316 1. RAPP. F.. Ann. Rec. Microhiol. (1966). 2. HUANG, A. S. and BALTIMORE. D., Nature talondon) 226, 325-327 (1970). 3. DOYLE, M. and HOLLAND, J. J.. Pm. Nat. Acad. Ski. USA 70, 2105-2108 (19X3). 4. COLE, C. N., SMOLEK, D., WIMMER, E. and BALTIMORE, D., J. Vim!. 7, 478-485 (1971). 5. COLE, C. N. and BALTIMORE, D.. J. Mol. Riol. 76, 325-343 (1973). 6. COLE, C. N. and BALTIMORE, D.. J. Mol. Hioi. 76, 345-361 ( 1973). 7. HOLLAND, cJ. J. and MCLAHEN. L. C., J. Ractrriol. 78, 596-597 (1959). 8. HALL, L. and RUEKEHT, R. R., Virologv 43, 152-165 (1971). 9. CHOPPIN, P. W.. Virology 39, 130-134 (1969). 10. PERRAULT, J. and HOLLANII, J. .J., Virology 50, 148-158 (1972).
60,579-583
(1974)
Defective
Interfering
Particles
and Vaccine LEROY
Reference
C. McLAREN
Department of Biology, Uniuersity of California Microbiology, I/niuersit.y of New Mexico
From Poliovirus
AND
JOHN
Strains J. HOLLAND
at San Diego, La Jolla, California School of Medicine, Albuquerque.
Accepted
April
Vaccine
92097 and Department New Mexico 87231
of
25. 1974
Defective interfering (DI) viral particles have been found to be associated with attenuated oral poliovirus vaccine and also with poliovirus vaccine reference strains. The DI particles replicate in low-passage human fibroblastic cells, the RSC-1 line of monkey kidney cells as well as in HeLa cells. DI particles of the attenuated polioviruses are generated after relatively few serial, high multiplicity passages of cloned, DI-free vaccine virus, whereas virulent strains of poliovirus generate a significant number of DI only after a large number of passages.
Defective interfering (DI) viral particles have been described for many classes of animal viruses (I, 2) and it has been suggested that they may play a significant role in viral diseases in uiuo (2). In support of this suggestion, it has recently been shown that defective T particles of vesicular stomatitis virus (VSV) provide protection of mice against viral disease and death following intracerebral inoculation of standard, infectious VSV (3). DI particles have been demonstrated by Cole and Baltimore using stocks of virulent type 1 poliovirus (Mahoney strain) (4). These DI particles were found to be generated after approximately 20 passages of virus at high multiplicity of infection of HeLa cells. They appear to be deletion mutants which lack about 15% of the RNA of standard virus and their density at equilibrium is lower in CsCl gradients. The DI particles contain the same capsid proteins as standard poliovirus, and they are efficiently adsorbed to HeLa cells and become uncoated. Being defective, they require coinfection of cells by standard virus to provide a helper-function for DI particle formation. In cells infected only by poliovirus DI particles, the capsid protein precursor (NCVP-l), capsid proteins and
therefore intact virions are absent, although other viral proteins encoded by the defective RNA genome are demonstrable (5, 6). In the present report, we show that DI particles are present in commercial preparations of oral poliovirus vaccine and also in poliovirus vaccine reference standards. The DI particles in attenuated poliovirus strains replicate in monkey kidney and human fibroblast cells as well as in HeLa cells. They appear to be generated more quickly in cells infected with attenuated poliovirus strains than with virulent type 1 poliovirus. BSC-1, and NHP (primary human skin fibroblasts, passage 7) cells were grown as monolayers in screw-cap glass bottles in Eagle’s medium containing 5% calf serum (MEM). Virulent poliovirus, type 1, was a clinical isolate previously typed by Dr. Edwin Lennette. Attenuated, vaccinereference strains of poliovirus, type 1 (TA3), type 2 (TB3) and type 3 (TC3) were kindly supplied by the Division of Biological Standards of the U.S.F.D.A. Trivalent, oral poliovaccine was from a commercial lot prepared by Wyeth Laboratories. For uridine labeling of viruses, approximately 2 x lo6 cells were infected at a multiplicity
580
SHORT
COMMUNICATIONS
of 100, and after adsorption at room temperature for 30 min, Eagle’s minimalessential-medium containing 2%) dialyzed calf serum, 5 pg of actinomycin D per ml and 200 PCi of [5-3H Juridine per ml was added. Following incubation at 37” for 4 hr radioactive media was removed, cells were rinsed with 0.15 M NaCl and then reincubated for 12 hr in MEM. Virus plaque assays were performed on HeLa cell monolayers under 0.4?~ agarose (7). Labeled polioviruses were purified by a modification of the method of Hall and Rueckert (8). Infected cells were disrupted by freezing and thawing five times and were then dialyzed overnight against 0.15 M NaCl containing lo- 3 M EDTA. Cell debris was then removed by centrifugation at 2000 R for 10 min. Nonlabeled, virulent, poliovirus, 10” PFU, was added as carrier, and the viruses were concentrated by centrifugation at 146,000 g for 90 min. Virus pellets were then suspended in 0.15 M NaCI, 0.2 M sodium phosphate pH 7.2 (PBS) and treated with ribonuclease, 10 pg per ml for 30 min at 25’ and further purified by gel filtration through Sepharose 2B (Pharmacia) columns equilibrated with PBS. Eluted viruses were then banded in CsCl (p = 1.33-1.34) by centrifugation at 140,000 R in a type 50 Ti rotor for 16 hr at 4”. Gradients were collected by puncturing the tubes at the bottom. Each fraction was precipitated by the addition of 10%~trichloracetic acid (TCA) and carrier (bovine serum albumin and yeast RNA, 25 pg of each per ml). Radioactivity in each TCA insoluble fraction collected was determined by dissolving the TCA precipitates in NCS tissue solubilizer (Amersham/ Searle) followed by the addition of Omnifluor (New England Nuclear). To determine the possible presence of DI particles in oral poliovaccine, we inoculated undiluted trivalent oral poliovaccine (0.2 ml) onto approximately lo6 HeLa cells. After 2 hr adsorption at 37” fresh medium was added and the culture incubated at 37”. After apparent cytopathic destruction of the cell monolayer undiluted cell culture fluid was reinoculated onto new HeLa monolayers and [3H]uridine labeled. The labeled, purified viral progeny was
found to contain a peak of radioactivity of similar density of that found for DI particles (Fig. 1). The densities of the virion and DI particle peaks were found to be 1.34 and 1.31, respectively, which is consistent with those reported for virulent type 1 poliovirus and its DI (4-6). Neutralization of more than 90% of the virus in the second HeLa cell passage of the vaccine virus was observed when reference type 1 poliovirus antiserum was employed in a plaque reduction assay. We next examined each of the three reference strains used fbr vaccine production. Reference strains, TA3, TB3 and TC3 of poliovirus types 1, 2 and 3 respectively, during second passage in HeLa cells, were labeled with [3H]uridine as described. Analysis of CsCl gradients for radioactivity revealed that each of the three reference viruses also generated DI particles as shown in Fig. 2. It will be noted that the second passage of TB2 in HeLa cells differs from TA3 and TC3 strains in that there was no significant amount of radioactivity in the virion peak. Infectivity assays on fractions collected from the p = 1.33-1.34 region of the gradient demonstrated little or no infectious virus was synthesized. This result was found not to be limited to strain TB3 and was also found occasionally with TA3 and TC3 in other experiments. This effect is
‘C
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I
-_ f
t
FIG. 1. Appearance ot’D1 particles associated with attenuated. oral poliovaccine. Second HeLa cell passage of vaccine virus, labeled with [“Hluridine was centrifuged at 146,000 p for 16 hr at 4’ in CsCi. p 1.93-l 34, (Beckman rotor Ti 501, collected into 0.3 ml fractions and analyzed for radioactivity. Higher density is at the left.
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FIG. 2. DI particles associated with poliovirus reference strains. A attenuated strain X3. Second HeLa cell passage of each virus was labeled with [3H]uridine Fig. 1.
likely related to the large ratio of DI to infectious virus particles in the inoculum resulting in interference as reported for virulent poliovirus (6). Analysis of virulent, type 1 poliovirus infected cells, included as a control with each experiment, consistently yielded a prominent virion and only a minor DI particle profile (Fig. 3). Because DI particles have thus far been described only for poliovirus replicating in HeLa cells, reference strains TA3, TB3 and TC3 of monkey kidney (MoK) cell origin were [3H]uridine labeled during second passage in either the BSC-1 line of MoK cells or in the low passage, fibroblasts. line NHP. DI particles were found to be produced by all three virus strains following infection of both BSC-1 and NHP cells. Fig. 4 illustrates the appearance of both virion and DI particle peaks in BSC-1 and NHP cells infected by TA3 virus and is similar to those observed previously for virus propagated in heteroploid HeLa cells. In addition, it was consistently observed that most of the radiolabel was present in the DI particle peak of virus-infected NHP cells. I n previous studies (4-6) it has been
*-
strain TA3; B strain TB8; C and analyzed as described in
t
CT ‘0 I
FE. 3. C&l density-gradient centrifugation ol virulent type 1 poliovirus, (3H]uridine labeled after 35 passages in HeLa cells. Gradient was analyzed as described in Fig. 1.
reported that a virulent strain of plaque purified type 1 poliovirus required 20~25 passages in HeLa cells before significant amounts of DI particle were apparent. Using our virulent strain of type 1 poliovirus only small quantities of labeled DI particles are generated in HeLa cells even after 90 serial passages. When we plaque-purified each of the poliovaccine reference strains the DI peaks were eliminated during the first few serial undiluted passages (see Fig. 5A). However,
582
SHORT
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t
n
i
\ , ..
fection of HeLa, BSC-1 or human diploid fibroblast cells. These DI showed the typical DI behavior of disappearing after cloning, and reappearing upon further serial high multiplicity passage. Using these plaque-purified poliovirus reference strains, it was clear that fewer serial passages were required for the appearance of DI particles than has been observed for virulent strains of poliovirus. It is conceivable that the relatively limited replication in vim of attenuated vaccine strains of poliovirus may be related to their apparent genetic potential for more rapid generation of DI particles. It is interesting that the diploid human fibroblasts generated a larger proportion of DI particles than did the HeLa or BSC-1 lines infected with the same virus pool (see Fig. 4). A similar influence of host cell type upon autointerference has been observed in other virus groups generating DI particles (2, 9. IO). There is presently no evidence regarding whether DI particles play any role in the ,7
FIG. 4. Production of DI particles of TA3 poliovirus in other cell lines. Both A: (HSC-1 cells) and B (NHP cell line) were [3H]uridine in the second passage of the reference virus cell type.
strain of infected labeled in each
using this plaque-purified virus, DI particles were found to be present after as few as five serial passages in HeLa cells. After a single passage in NHP cells of the HeLa passage 5, plaque-purified TA3 virus, most of the radiolabel was found to be in the DI particle peak as shown in Fig. 5B. The generation of DI particles by virulent strains of type 1 poliovirus has been shown to require many successive passages of virus at high multiplicity (4). It is therefore not surprising that such particles might be present in attenuated strains of poliovirus which have undergone many passages in cell culture prior to their use in vaccine preparation. Our analysis of commercially distributed oral poliovaccine prepared in monkey kidney cell cultures, as well as reference strains of types 1, 2 and 3 poliovirus shows that significant amounts of DI particles are demonstrable after in-
‘,
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I
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.
I
._.;
:
.._...
..
_ : ;.
-. ‘.
/ j
a
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”
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.:
. .
. . . . .I 1,
’ /,.,
:_
3,
.,
FIG. 5. Dl particles associated with plaque purified TA3 strain poliovirus. (A) Plaque purified virus radiolabeled after infection of NHP cells, following one passage in HeLa cells; (B) plaque purified virus after five serial passages in HeLa cells and a single passage in NHP cells. Infected cells were [3H]uridine labeled and centrifuged in CsCl as previously described.
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attenuation of vaccine strains, or in the production of immunity following ingestion of attenuated, oral poliovaccine. It is noteworthy that defective T particles protect mice prophylactically against lethal infection following intracerebral inoculation of VSV. Mice inoculated with virus-free, T particles of WV have also been shown to develop immunity to challenge 10 days later with homologous virus (3). It would be informative to determine whether poliovirus vaccine strains are more virulent in the intraspinal monkey test after they are freed of DI particles by cloning, and, if so, whether addition of purified DI particles to the cloned virus restores attenuation. ACKNOWLEDGMENTS We thank Estelle Bussey for excellent technical assistance, and Dr. Michael Doyle for helpful discus
sions. Supported by Grant tional Cancer Institute.
CA 10802 from the Na-
REFERENCES 23. 293 316 1. RAPP. F.. Ann. Rec. Microhiol. (1966). 2. HUANG, A. S. and BALTIMORE. D., Nature talondon) 226, 325-327 (1970). 3. DOYLE, M. and HOLLAND, J. J.. Pm. Nat. Acad. Ski. USA 70, 2105-2108 (19X3). 4. COLE, C. N., SMOLEK, D., WIMMER, E. and BALTIMORE, D., J. Vim!. 7, 478-485 (1971). 5. COLE, C. N. and BALTIMORE, D.. J. Mol. Riol. 76, 325-343 (1973). 6. COLE, C. N. and BALTIMORE, D.. J. Mol. Hioi. 76, 345-361 ( 1973). 7. HOLLAND, cJ. J. and MCLAHEN. L. C., J. Ractrriol. 78, 596-597 (1959). 8. HALL, L. and RUEKEHT, R. R., Virologv 43, 152-165 (1971). 9. CHOPPIN, P. W.. Virology 39, 130-134 (1969). 10. PERRAULT, J. and HOLLANII, J. .J., Virology 50, 148-158 (1972).