430
DISCUSSION
AND PRELIMINARY
different types. With FMDV, types 0 and A, it has been shown that the 20-mp components have nothing in common antigenically with each other or with other components of both types. The 7-rnp components show only a partial mutual antigenic community. REFERENCES 1. CROWLE,A. J., “Immunodiffusion,”
p. 70. Acadcmic Press, New York, 1961. 2. BROWN, F., and CRICK J., Virology 5, 133 (1958); J. Immunol. 82,444 (1959). S. VAN Oss, C. J., and HECK, Y. S. L., 2. Immunitiitsforsch. 122, 44 (1961); Rev. Immunol. 27 (1963). C. J. VAN Oss’ L~ONE DHENNIN LOUIS DHENNIN Laboratory of Physical Biochemistry, I.N.R.A. &ole Nationale Ve’te’rinaire d’Alfort, and Virus Department, Laboratoire cherches Ve’te’rinaires Alfort (Seine), France Accepted December 3,196s
Central
de Re-
1Present address: Department of Microbiology and Immunology, Montefiore Hospital, New York City. Inhibition
of the Multiplication
of Pseudorabies
Virus by Cyclohexamide
Cycloheximide (1) is an antibiotic isolated from cultures of Streptomyces griseus (2). The compound possesses activity against a broad spectrum of fungi (3, 4), inhibits certain algae (5) and a protozoan (6) , but is relatively inactive against a number of species of bacteria (S,4). Some interest has been shown in cycloheximide for cancer chemotherapy as a result of marginal activity against certain experimental neoplasms (7, 8). When tested for antiviral activity in this laboratory, cycloheximide was found to inhibit formation of plaques by all viruses tested using an agar diffusion method (9) at 10 pg or less per cellulose disk (Table 1). No cytotoxicity is evident at these concentrations by microscopic examination of cell monolayers. The nature of the inhibition has been examined in rabbit kidney cells infected with pseudorabies virus, Aujeszky strain.
REPORTS
Cells of monolayer cultures, prepared from rabbit kidney (10) were dispersed with trypsin. The cells were suspended at lo6 cells per milliliter in Earle’s saline with 0.5% lactalbumin hydrolyzate and 6% horse serum. Virus was added at a ratio of 10 plaque-forming units (PFU) per cell, and the suspension was gently agitated. After 20 minutes (0 time) the cells were sedimented by cent(rifugation at 1000 g for 5 minutes and the medium was removed. The cells were washed twice with 15 ml of medium. They were then resuspended in medium at 10” cells per milliliter, and incubated at 37”. At intervals after infection 2-ml aliquots were removed from the culture and the free virus content was determined by plaque titration. Both rabbit kidney and chick embryo cell monolayers were used for assay; the titers were comparable on both host cells. Cycloheximide from a concentrated solution was added to the culture at various times. The compound was removed during the course of an experiment by sedimenting the cells and resuspending them in fresh medium. One microgram per milliliter is the maximum well-tolerated concentration of cycloheximide for the maintenance of rabbit kidney cells on glass over a 5-day period as determined by cell count and microscopic examination. The characteristics of a single-step multiplication cycle of pseudorabies in rabbit TABLE 1 VIRUSES SENSITIVE TO CYCLOHEXI>~IUEAT 10 pg PER DISK IN AN AG.~R DIFFUSION TEST AS DETERMINED BY PL.~QUE IN~IIBITION Virus
Source of host cells
Coxsackie, Bl, Conn. 5 Herpes simplex, HF Influenza A, WSN Influenza B, GL Mouse hepatitis, MHV3 Newcastle disease, Bonney Polio, type 2, Lansing Pseudorabies, Aujeszky Rous sarcoma, C.T.583” Semliki Forest Vaccinia, WR Yellow fever, 17D
HELA Rabbit kidney Chick embryo Chick embryo Mouse embryo Chick embryo HeLa Rabbit kidney Chick embryo Chick embryo Chick embryo Chick embryo
a Tumor foci inhihited.
DISCUSSION AND PRELIMINARY
REPORTS
431
of rabbit kidney cells obtained under the above con- t,ime, inhibits t’he multiplication kidney cells at the concentration used (1 ditions are shown in Fig. 1. When cycloheximide at 1 pg/ml is added at any time pg/ml). No antiviral effect is obtained at a up to 4 hours after infection, inhibition of concentration which does not inhibit cell multiplication (0.1 pg/ml). Thus, the inhivirus multiplication is practically complete. bition observed is not specific for virus synLater addition (7 hours) of cycloheximide is without effect. When cycloheximide is thetic processes. It is plausible to ascribe added prior to 4 hours after infection and the antiviral action of the compound to an removed at a later time, virus production is inhibition of protein and DNA synthesis on delayed only by the same period of time the the basis that these were the initial alteratreatment had lasted. This is illustrated in, tions evident in the metabolism of SacchaFig. 1 for a 6-hour treatment starting at 0 romyces carlsbergensis (11) and HeLa cells (R. F. Haff, to be published) treated with time. Supplementary studies indicate that cy- cycloheximide. Such an effect should proof virus multiplication at cloheximide is not virucidal, nor does it in- duce inhibition any time prior to, but not necessarily influence virus release from the cell. Inhibition similar to that shown in Fig. 1 is observed cluding, the assembly stage, as was indeed when the virus content of the infected cells, observed. ACKNOJVLEDGMENT rather than that of the medium, is measured. It is concluded, therefore, that cycloheximThe author wishes to express appreciation to Dr. C. E. Hoffmann fcr assistance in the preparaide inhibits synthesis of virus during the inition of this manuscript. tial four-fifths of the latent period at least; later stages of viral replication are refracREFERENCES tory. Furthermore, the inhibition may be ac1. KORNFELD,E. C., *JONES,R. G., and PARICE, complished throughout the early stages of T. V., J. Am. Chem. Sot. 71, 150-159 (1949). synthesis, since treatment for the initial 6 2. LEACH, B. E., FORD,J. H., and WHIFFEN, A. J , hours of infection delays the rise in virus J. Am. Chem. Sot. 69, 474 (1947). titer by a comparable interval. 3. WHIFFEN, A. J., BOHONAS,N., and EMERSOS, These findings may be placed in proper R. L., J. Bacterial. 52, 610-611 (1946). perspective by the observation that cyclo4. PHILLIPS, G. B., and HANEL, E., JR., J. b’acheximide, although permitting maintenance teriol. 60, 104105 (1950). of viable cells for an extended period of 6. HUNTER, E. O., JR., and MCVEIGH, I., Am. J. Botany 48, 179-185 (1961). 6. MEFFERD,R. B., JR., and LOEFER,J. B., Physiol. 2001. 27, 115-11s (1954). 7. BATEMAN,J. C., and KLOPP, C. T., Proc. Am. Assoc. Cancer Res. 1, 3 (1953). 8. REILLY, H. C., STOCK,C. C., BUCKLEY, S. M., and CLARK, D. A., Cancer Res. 13, 684-687 x CONTROL 0 CYCLOHEXIMIOE ADDED AFTER 4hrs. . CYCLOHEXIMIDE ADDED AFTER q
TlME
AFTER
7hrs.
CYCLOHEXIMIDE REMOVED AFTER
INFECTION
- HOURS
FIG. 1. Effect of 1 pg cycloheximide per milliliter on the multiplication of pseudorabies virus in rabbit kidney cells under conditions of a single-step multiplication cycle.
(1953).
9. DESOMER,P., and PRINZIE, A., Virology 4, 387388 ( 1957). 10. KAPLAN, -A. S., Virology 4, 435457 (1957). 11. KERRIDGE,D., J. Gen. Mio~obiol. 19, 497-506 (1958). RICHARD F. HAFF’ Stine Laboratory Industrial and Biochemicals Department E. I. du Pont de Nemours and Co. Neusark, Delaware Accepted December 19, 1963
1Present address : Research and Development Division, Smith Kline and French Laborat,ories, Philadelphia 1, Pennsylvania.