APPLiED MicRosioLoGY, Nov. 1969, p.

897-900 Copyright ) 1969 American Society for Microbiology

Vol. 18, No. 5 Printed in U.S.A.

Accelerated Plaque Formation by Fowlpox Virus in the Presence of Chymotrypsin

BONNIE B. ASCH1 AND GEORGE E. GIFFORD Department of Microbiology, University of Florida College of Medicine, Gainesville, Florida 32601

Received for publication 18 July 1969

Chymotrypsin enhanced fowlpox virus plaque formation in chick embryo cell cultures. A simplified plaque assay for fowlpox virus is described. Plaques were produced in 3 days when chymotrypsin was included in a serum-free fluid overlay. Plaques were also produced in 5 to 6 days under an agar overlay when a medium containing fetal calf serum was employed. Kinetics of plaque formation were also studied, and it was shown that fowlpox virus plaque diameters grow at a linear rate.
This study was undertaken as a consequence of the results obtained by Gifford and Klapper (3, 4) who demonstrated that the number and size of vaccinia virus plaques were increased in the presence of trypsin or chymotrypsin. We wanted to see whether the phenomenon could be extended to other poxviruses. Fowipox virus replicates slowly in chick embryo cell cultures, and visible plaques are produced 8 to 11 days after infection (7). The studies presented here show that when chymotrypsin was added to chick embryo cell cultures infected with fowlpox virus, plaques appeared in 3 days. The phenomenon was studied to develop a rapid plaque assay for this virus.
MATERIALS AND METHODS Cell cultures. Primary cell cultures were established from eviscerated and decapitated 10- to 11-day-old chick embryos by using techniques previously described (5). Square bottomed, 2-oz, soft-glass bottles (3 by 3 by 6 cm) were each seeded with 12 X 106 cells in a volume of 5 ml of growth medium. A monolayer of about 3 million cells was usually found within 48 to 72 hr after incubation at 37 C. Media. The growth medium for the establishment of the cell cultures consisted of Gey's balanced salt solution (BSS) supplemented with 0.06% sodium bicarbonate, 5% calf serum, 0.1% lactalbumin hydrolysate, and 0.1% proteose peptone. Maintenance medium was similar to the growth medium except that 0.1% yeast extract replaced the calf serum. Agar overlay medium consisted of the growth medium but with 7.5% fetal calf serum replacing the calf serum and 0.4% "lonagar no. 2" added. Potassium penicillin G and streptomycin sulfate at 250 units and 125 ug, respectively, were added to all media. Virus. Fowlpox virus was a vaccine strain (list no.
I Present address: Department of Surgery, The University of Texas Southwestern Medical School, Dallas, Tex. 75235.

5179) obtained from the Amdal Company, Agricultural Division of Abbott Laboratories, North Chicago, Ill. Progeny virus was derived by inoculating the chorioallantois of developing chick embryos with 0.1 ml of a 1:10 dilution of the reconstituted vaccine and incubating the eggs at 37 C. After 48 hr, the chorioallantois were removed, frozen, and then homogenized in maintenance medium with a mortar and pestle. After centrifuging at 480 X g for 30 min to remove tissue debris, the virus-containing supernatant fluid was dispensed into glass ampules and stored at -60 C. Chymotrypsin. A purified, crystalline a-chymotrypsin (Worthington Biochemical Corporation, Freehold, N.J.) was used. The enzyme was reconstituted with sterile distilled water to give a concentration of 1 mg/ml, dispensed in samples of 1 ml, and stored at -20 C. Dilutions were made in maintenance medium as needed to yield 1.5 to 5 jsg/ml when added to cell cultures.

RESULTS Plaque assay of fowlpox virus with a fluid overlay containing chymotrypsin in maintance medium. The assay system is essentially the procedure described for vaccinia virus (3, 4). Virus and different concentrations of chymotrypsin, in maintenance medium, were added to welldrained plaque bottles in a total final volume of 2.0 ml. Cultures were incubated for various time periods at 37 C. The medium was then decanted and the cell sheets were stained with crystal violet. The addition of 1.5 to 5 jsg of chymotrypsin per ml to fowlpox virus in serum-free maintenance medium resulted in the appearance of plaques within 3 days after inoculation into chick embryo cell cultures. The concentration of chymotrypsin employed in this procedure proved to be critical. If the concentration of chymotrypsin in the fluid overlay was greater than 5 ,ug/ml

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(10 jug/culture), too many cells were detached from the glass and plaques could not be discerned. If less than 1.5 MAg/ml was employed, the effect of chymotrypsin on plaque formation in 3 days was lost. Figure 1 shows the appearance of plaques 3 days after infection. Kinetics of plaque formation by fowipox virus. This study was done to determine the rate of appearance of plaques and the time needed for all plaques to become visible. Chick embryo cell cultures were infected with fowlpox virus and the virus was allowed to adsorb for 1.5 hr at room temperature. The monolayers were then overlayed with 2 ml of maintenance medium containing chymotrypsin. The bottles were incubated at 37 C and sample bottles were removed at various time intervals. After staining with crystal violet, the plaque images were drawn with the aid of a photographic enlarger and then were counted and measured. Visible plaques began to appear 44 to 46 hr after infection, with maximal number of plaques being reached at about 78 hr (Fig. 2).

The average diameter of the plaques continued to increase linearly as long as they were observed (Fig. 3). Apparently, there was no maximum size for plaques as long as the cell layer was intact. The rate of growth of the plaque diameter was 0.031 mm/hr. Secondary plaques were not seen over the time interval studied. This linearity of plaque growth has previously been reported for vaccinia virus (6). Growth of fowlpox virus in the presence and in the absence of chymotrypsin. These studies were conducted to compare the dynamics of replication of fowlpox virus in chick embryo cell cultures in the presence and in the absence of chymotrypsin. Chick embryo cell cultures in plaque bottles were prepared as previously described and, at the time of use, were found to contain approximately 2.3 x 106 cells per culture. Fowlpox virus was allowed to adsorb for 3 hr at room temperature with or without chymotrypsin. A resultant multiplicity of 2.4 plaque-forming units (PFU)/cell was obtained with both conditions. The cell

FIG. 1. Appearance offowipox virus plaques in chick embryo cell cultures. The culture on the left was stained with crystal violet 3 days after infection and employed a fluid overlay with chymotrypsin as described in the text. The culture on the right shows plaques 6 days after infection under an agar-overlay. The arrows point to "ring-like" areas described by Wittman and Mayr (11).

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represents the mean of three to five bottles. A faximum number of plaques is obtained approximatejly 78 hr after infection.
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monolayers were washed twice with maintenance medium and were overlaid with 2 ml of maintenance medium or 2 ml of maintenance medium containing 5 ,ug of chymotrypsin per ml. Cultures were incubated at 37 C, and sample cultures 80were removed at various time intervals thereafter and stored at -60 C. The cultures were subsequently thawed and frozen and thawed three times, and the virus was assayed by the procedure outlined above using chymotrypsin in the fluid 60overlay. The two growth curves are shown in Fig. 4, and both curves have similar shapes and slopes but are separated by about 1 day. Since the displacement is apparent soon after infection, chymotrypsin may have enhanced some early event in the replication cycle. Plaque assay of fowlpox virus in cultures with 30an agar overlay. Since the presence of a proteolytic enzyme might interfere with certain 20experiments, studies were undertaken to investigate the production of plaques by fowlpox virus in chick embryo cell cultures with an agaroverlay system. Several combinations of various media with "Ionagar" were tested and observed r----, ,up to 6 days after infection with virus. The best 40II 50 60 770 90 go ' medium for plaque formation was growth meHOURS AFTER INFECTION dium with 7.5% fetal calf serum in which plaques FIG. 2. Percentage of primary plaques a at different times after infection in the prese nppeaofng appeared in 5 to 6 days. The appearance of the chymotrypsin-containing fluid overlay. Eacch Point plaques under the agar overlay is shown in
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FIG. 3. Growth of plaque diameters with time in cultures with a fluid overlay containing 3 mg of chymotrypsin per ml. Plaque diameters were measured on projected images magnified 6.5 times. Rate of increase of plaque diameters was approximately 0.031 mm/hr.

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FIG. 4. Growth curves offowlpox virus in the presence and absence of chymotrypsin. Multiplicity of infection was 2.4 PFU/cell.

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Fig. 1. Ringlike areas with intensely staining perimeters and lightly staining centers were found in these cultures (Fig. 1). The rate of increase of plaque diameters was 0.025 mm/hr, and these plaques also grew linearly. DISCUSSION Enhancement of plaque size and number resulting from the incorporation of proteolytic enzymes into the overlay medium has been described for certain enteroviruses (10), reoviruses (9), myxoviruses (1), and vaccinia (3, 4). The present study shows that plaque formation by fowlpox virus is greatly accelerated by the presence of chymotrypsin in the fluid overlay. Mayr and Kalcher (7) found that fowlpox virus produced plaques on chick embryo cell cultures. Development of plaques was slow, and visible plaques could not be seen until about 8 days after infection; 11 days were required before all of the plaques became visible. Feeding of the cells at 3-day intervals was necessary to maintain the cells for the long period of time under agar. These authors also found that fowlpox virus plaque formation was very sensitive to the density of the cell layer. They reported larger and more distinct plaques formed in petri-dish cultures prepared with 15 million cells than with 24 million. The impression was obtained that the plaques developed earlier in the lighter cultures. Cultures prepared in our laboratory apparently consisted of fewer cells, which may have contributed to the fact that we found a maximum number of plaques under agar in 5 to 6 days after infection. Considering the surface area employed, cultures in our laboratory would have been prepared with about half as many cells as they employed in their lightest cultures. In the agar-overlay system, circular areas about the size of a plaque were sometimes found that stained more intensely than the rest of the cell monolayer. Another form that was found was similar in appearance to those described by Wittman and Mayr (11) as "Ringzonenbildung," i.e., ring-like areas which have an intensely staining perimeter with a lightly staining center. These ring zones have been described on both chorioallantoic membranes of developing chick embryos and in cell cultures (7, 8, 11), and the authors

conclude that these areas are formed by hyperplasia of cells infected with the virus. These cells were later seen to undergo lysis and thus finally form a plaque or lesion. After this study was completed, a study by Gafford, Sinclair, and Randall (2) was published which showed plaque formation in chick embryo cells in 6 to 7 days. Their virus growth curves showed that the increase in infectious virus was exponential from 36 to 72 hr and that maximum virus was synthesized by 72 to 96 hr. Growth of fowlpox virus was enhanced by the presence of mycoplasma. It is possible that mycoplasma may produce low levels of proteolytic enzymes or, conversely, that proteolytic enzymes may activate low levels of mycoplasma contaminants.
ACKNOWLEDGMENT This investigation was supported by Public Health Service research grant AL-04361 from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED 1. Came, P. E., A. Pascale, and G. Shimonaski. 1968. Influenza plaque enhancement by pancreatin. Arch. Gesamte. Virusforsch. 23:346-352. 2. Gafford, L. G., F. Sinclair, and C. C. Randall. 1969. Growth cycle of fowlpox virus and change in plaque morphology and cytopathology by contaminating mycoplasma. Virology 37:464-472. 3. Gifford, G. E., and D. G. Klapper. 1967. Enhancement of vaccinia virus plaque formation by trypsin. Proc. Soc. Exp. Biol. Med. 126:515-517. 4. Gifford, G. E., and D. G. Klapper. 1969. Effect of proteolytic enzymes on vaccinia virus replication. Arch. Gesamte. Virusforsch. 26:321-333. 5. Gifford, G. E., M. V. Mussett, and E. Heller. 1964. Comparative studies on the production and assay of interferon. J. Gen. Microbiol. 34:475-481. 6. Lindenmana, J., and G. E. Gifford. 1963. Studies on vaccinia virus plaque formation and its inhibition by interferon. L Dynamics of plaque formation by vaccinia virus. Virology 19:283-293. 7. Mayr, A., and K. Kalcher. 1961. Plaque-Bildung bei den Geflugelpockenviren. Arch. Gesamte. Virusforsch. 11: 307-325. 8. Mayr, A., and G. Wittman. 1957. Observations on local spread of pox viruses in tissue. Science 125:1034-1035. 9. Wallis, C., J. L. Melnick, and F. Rapp. 1966. Effects of pancreatin on the growth of reovirus. J. Bacteriol. 92:155160. 10. Wallis, C., F. Morales, J. Powell, and J. L. Melnick. 1966. Plaque enhancement of enteroviruses by magnesium chloride, cysteine, and pancreatin. J. Bacteriol. 91:19321935. 11. Wittman, G., and A. Mayr. 1956. Zur Ringzonenbildung in Virus-infizierten tierischen Geweben. Zb. Vet. Med. 3:641642.