A macrofocus assay for Rous sarcoma virus (RSV) in chicken embryo cells

DISCUSSION

AND

PRELIMINARY

findings support the contention that the particles observed in Fig. 1 are the LDH agent. Although the LDH agent seemed to be essentially spheroidal in shape (Figs. 1 and 2), about 20% of the particles showed some degree of pleomorphism, including projections of the types seen in Figs. 3-5. Further studies revealed that if the resuspended virus was not fixed with osmium tetroxide, but instead was stained directly with PTA, an even larger percentage of the particles (over 80%) showed pleomorphic forms, including protrusions of the types seen in Figs. 6 and 7. These findings are of particular interest in the light of recent work which indicates that certain animal viruses, Moloney leukemia agent (7), Rauscher virus (8), and equine abortion virus (9), may have a tail. However, there is also considerable evidence in the literature which indicates that the pleomorphic forms and the projections found with a number of other animal viruses, avian myeloblastosis (IO), Rous sarcoma (II), fowl plague (12), and Newcastle disease (1%15), are artifacts. The projections on the LDH agent point to the possible existence of a tail on this virus. However, the variation in shape and size of these projections and the greater number of pleomorphic forms in the unfixed material suggest even more strongly that they are artifacts produced in the preparation and staining of the virus. Further studies designed to critically evaluate the significance of these projections seem warranted. ACKNOWLEDGMENT The authors gratefully acknowledge the many helpful discussions with Dr. Albert J. Dalton during the course of this work. REFERENCES 1. RILEY, V., LILLY, F., HUERTO, E., and BARDELL, D., Science 132,545-547 (1960). b. NOTKINS, A. L., and SCHEELE, C., Virology 20, 640-642 (1963). 3. NOTKINS, A. L., and SHOCHAT, S. J., J. Exptl. Med. 117,735-747 (1963). 4. RILEY, V., Proc. Sot. Exptl. Biol. Med. 104,

751-754 (1960). 5. PALADE, G. E., J. Exptl. (1952).

Med.

95,

285-298

271

REPORTS

6. BRENNER, S., and HORNE, R. W., Biochem. Biophys. Acta 34, 103-110 (1959). 7. DALTON, A. J., HAGUENAU, F., and MOLONEY. J. B., J. Natl. Cancer Inst. 29, 1177-1179 (1962). 8. ZEIGEL, R. F., and RAUSCHER, F. J., J. Natl. Cancer Inst. 30,207-219 (1963). 9. SHARP, D. G. and BRACKEN, E. C., Virology 10, 419-431 (1960). 10. BONAR, R. A., HEINE, U., BEARD, D., and BEARD, J. W., J. Natl. Cancer Inst. 30, 949997 (1963). 11. DOURMASHKIN, R. R., and SIMONS, P. J., J. Ultrastmct. Res. 5,505-522 (1961). 12. WATERSON, A. P., ROTT, R., and SCHAFER, W., 2. Naturforsch. 16b, 154-156 (1961). 13. BANG, F. B., J. Exptl. Med. 88, 251-266 (1948). 14. ELFORD, W. J., CHU, C. M., DAWSON, I. M., DUDGEON, J. A., FULTON, F., and SMILES, J., Brit. J. Exptl. Pathol. 29, 59S599 (1948). 15. SHARP, D. G., ECKERT, E. A., BEARD, D., and BEARD, J. W., J. Bacterial. 63, 151-161 (1952). HOWARD A. BLADEN, JR. ABNER LOUIS NOTKINS of Histology and Pathology, and Laboratory Laboratory of Microbiology National Institute of Dental Research National Institutes of Health Bethesda 14, Maryland Accepted July 24, 1963

A Macrofocus

Assay

for

(RSV) in Chicken

Rous Sarcoma Embryo

Virus

Cells’

Manaker and Group& (1) observed that discrete foci of altered cells were formed in monolayer tube cultures of chicken embryo cells after exposure to RSV and that the number of foci per culture was directly related to the concentration of virus inoculated. These observations were extended by Temin and Rubin (2)) who added an agar overlay to petri dish cultures of cells in order to minimize the chance of formation of secondary foci. A 7-day macroscopic assay for RSV in second-passage chicken embryo cells cultured in tubes is described here. After an incubation period of 7 days cells, at 40”, macrofoci of virus-altered clearly visible to the naked eye, were formed. This higher t,emperature of incuba‘This investigation was supported by the National Cancer Institute, National Institutes of Health, United States Public Health Service.

272

DISCUSSION

AND

FIG. 1. Culture of chicken embryo cells infected with approximately 40 focus-forming units (FFU). The agar overlay was removed and the culture was stained with neutral red 7 days after infection. Magnification : X 1.6.

TABLE TYPICAL

Dilution of RSV

1

TITRaTIONS OF STANDARD IN CHICKEN EMBRYO CELLS

RSV

Number of macrofoci per tubea Expt. 1

Expt. 2

Expt. 3

Expt. 4

10-d C c C C 10-s NC 66, 60, 74 NC NC lo-6 23, 4, 21 13, 8, 15 32, 27, 28 12, 21, 19 10-T 2, 5, 4 2, 0, 1 4, 2, 10 2, 3, 3 Control 0, 0, 0 0, 0, 0 0, 0, 0 0, 0, 0 ---L-------__------ED&n1

10s .2

0 C, Confluent

108 .i

macrofoci;

10s .4

108.2

NC, not counted.

tion was found to be critical for the formation of macrofoci. The assay system was reproducible, and the relationship between the number of macrofoci and the infective titer of the inoculum was linear. Since the macrofoci were large enough to be counted with the naked eye, the time consumed in microscopic counting, and its inherent errors were eliminated. Rubber-lined screwcap tubes were used to eliminate the necessity of COP incubation or the incorporation of special buffers. The standard growth medium (SGM) consisted of the following: Scherer’s maintenance solution, 68% ; tryptose phosphate broth (Difco), 20%; heat inactivated (56” for 30 minutes) calf serum, 10%; beef embryo extract, 2.0%; penicillin G, 100 units/ ml; streptomycin, 100 pg/ml; pH adjusted to 7.4 with 7.5% sodium bicarbonate solu-

PRELIMINARY

REPORTS

tion. The agar overlay medium consisted of: Solution A, which was the same as SG?VI with the exception that, it contained 68% double-strength Scherer’s maintenance solution; and Solut’ion B, which consisted of 1.6% Noble’s agar (Difco) in distilled water. Equal amount,s of Solution A (warmed to 45”) and Solution B (melted and cooled to 45”) were mixed. The mixture was held at 45” in a water bath. Standard frozen RSV, stored at -7O”, was provided by the National Cancer Institute. Eggs were obtained from Shamrock Farms, North Brunswick, New Jersey. Twelve-day-old chicken embryos were pooled, minced, washed twice in Hanks’ salt solution, and trypsinized (0.25% trypsin, Difco, in Hanks’ salt solution) by agitation with a magnetic stirrer for 1 hour at 37”. The cells were then washed 3 times in cold Hanks’ salt solution, filtered through 3 layers of sterile gauze, suspended in SGM, and counted. Sixteenounce rubber-stoppered bottles were seeded with 20 ml of cells (500,000 cells/ml) in SGM and incubated for 3 days at 37”. These primary cultures were then washed once with Hanks’ salt solution and the cells were removed from the glass with 0.125% trypsin in Hanks’ salt solution. Tubes (16 x 150 mm) were then seeded with 200,000 cells contained in 1.0 ml of SGM and incubated for 24 hours at 37”. Twenty-four hours after seeding, the media were decanted and the cultures were infected with O.l-ml amounts of virus diluted in SGM, incubated for 1 hour at 37”, overlaid with 2.0 ml of agar medium; the cultures were then incubated at 40” in a stationary slanted position (5’). At 3 days and again at 5 days after infection, all cultures were fed by adding 2.0 ml of agar medium on t.op of the original agar layer. This feeding schedule was found to be essential. Seven days after infection, the agar was removed by inverting and shaking the tubes, and 1.0 ml of a 1: 30,000 dilution of neutral red in Hanks’ salt solution was added to each culture tube. The cultures were then incubated for l-2 hours at 37”; the deeply stained macrofoci were then counted without the aid of magnification. The tubes were examined against a white background under a bright light’.

DISCUSSION

AND PRELIMINARY

After 7 days of incubation at 40”, the size of the macrofoci ranged from 0.25 to 1.0 mm in diameter and were clearly visible to the naked eye. After staining with neutral red, the macrofoci were stained dark red in marked contrast to the background of normal cells (Fig. 1). Approximately 2-3 days after infection, small discrete groups of refractile, rounded cells appeared which upon further incubation developed into large multilayered masses of cells, or macrofoci. The morphological changes observed were identical to those described previously (1, 2). In contrast to uninfected control cultures, there was a marked increase in production of acid during the development of the macrofoci. A series of titrations of standard RSV were carried out at weekly intervals in order to determine the sensitivity and reproducibility of the assay. In each titration, standard RSV was titrated in secondary cultures of cells obtained from groups of 5 pooled chicken embryos. Triplicate cultures were infected with serial tenfold dilutions of the virus, and the number of macrofoci per tube was counted and used to determine the infective titer (EDS,,) per milliliter. These data are summarized in Table 1; they indicate that the assay was reproducible and that a proportional relationship existed between concentration of virus and the number of macrofoci produced. It should also be noted that, under the conditions described, at no time were cell cultures found to vary in susceptibility to RSV. Indeed, the EDbO per milliliter of standard RSV in 2nd, 3rd, 4th, and 5th passage cells was found to be 1Os.4, 108.4, 108.2, and 10*.3, respectively. REFERENCES I. MANAKER, R. A., and GROUP& V., Virology 2, 838-840 ( 1956). 2. TEMIN, H. M., and RUBIN, H., Virology 6, 66% 688 (1958). EUGENE V. ADAMS’ VINCENT GROUP% Institute of Microbiology Rutgers, the State University New Bruswick, New Jersey Accepted July 8,196s “Squibb

Predoctoral

Fellow.

REPORTS

Precipitation from

and

Solutions

273

Recovery of

Low

of Ionic

Sindbis

Virus

Strength’

Taylor et al. (1) found that purified preparations of Eastern equine encephalitis virus precipitated when dialyzed against distilled water. We have studied the solubility of another purified arborvirus, Sindbis virus, and found that it can be precipitated simply by dilution to a low ionic strength. Methods for the purification and titration of Sindbis virus labeled with P32 have been described (2). These methods were modified by using the following solution for suspending the viral pellets after the ultracentrifugations: NaCl, 0.176 M; MgCIZ , 0.002 M; sodium phosphate buffer, pH 7.0, 0.001 M (ionic strength, 0.185). The final viral suspension contained 4 x 1012 PFU/ ml, 7700 cpm/ml, nearly equally divided between RNA and phospholipid, and 1.2 mg protein/ml, as measured by the FolinCiocalteu method (S). Volumes of 50 pl of this suspension were placed in small conical tubes and diluted with 0.5-ml of solutions containing 0.002 M MgCls , 0.001 M sodium phosphate buffer, pH 7.0, and sufficient NaCl to give the desired final ionic strengths. After 14 hours at 4”, the solutions of low ionic strengths contained a fluffy, white, microscopically amorphous precipitate. Each tube was centrifuged at 5000 g for 10 minutes, and the supernatant fluids were analyzed for P32 and protein. The results are shown in Table 1. Sindbis virus was completely insoluble at an ionic strength of 0.03. The titer of the supernatant fluid from the tube with an ionic strength of 0.03 was less than lo7 PFU/ml compared with 3.7 X loll PFU/ml in the tube of ionic strength 0.15, which had no precipitate. When the precipitate from the solution of ionic strength 0.03 was redissolved in 0.55 ml of the original virus suspending medium, the radioactivity and protein were completely 1 This investigation was supported in whole by United States Public Health Service Research Grant AI 04531-02 from the Institute of Allergy and Infectious Diseases.