Plaque Formation by Sendai Virus of Parainfluenza Virus Group, Type 1. on Monkey, Calf Kidney and Chick Embryo Cell Monolayers

Japan. J. Microbiol. Vol. 15 (2), 175-183, 1971 Plaque Formation by Sendai Virus of Parainfluenza Virus Group, Type 1. on Monkey, Calf Kidney and Chick Embryo Cell Monolayers Hiroshi SHIBUTA, Masayuki AKAMI, and Minoru MATUMOTO Institute of Medical Science, University of Tokyo, Tokyo (Received for publication, November 9, 1970) ABSTRACT Cynomolgus monkey kidney cells were found to be a highly sensitive medium for plaque formation of Sendai virus and provided a practical method for the infectivity assay of the virus. The method gave reproducible titers and was as sensitive as the egg inoculation method and was simple enough to be used routinely. Sendai virus formed no recognizable plaques on chick embryo cell monolayers. However, the incorporation of a small amount of trypsin into the overlay medium induced plaques. This method gave comparable titers to those obtained on monkey kidney monolayers. Sendai virus formed plaques on calf kidney cell monolayers, but difficulties in the application of the method to infectivity assay arose from the fact that the plaques formed in calf kidney cells were small and not homogeneous in size and were not clearly defined. Sendai virus or HVJ (hemagglutinating virus of Japan) classified with the HA2 virus as parainfluenza virus type 1, can be assayed by inoculation into embryonated eggs or tube cultures of monkey or calf kidney cells, using hemagglutinin production as a criterion for infection. These methods are highly sensitive, but for a more reliable and simple quantitation of the virus, a plaque count method is desirable. This technique also lends itself to the isolation of viral clones. Certain Sendai virus strains were reported to form plaques on kidney cell cultures of newly hatched chicks [14] and calves [11]. We tested monolayer cultures of monkey and calf kidney cells and chick embryo cells, and found monkey kidney monolayers highly sensitive for plaque formation by Sendai virus. This method was readily adapted to the infectivity assay of the virus. Calf kidney cell cultures, however, were shown to be less suitable for plaque assay because of lower plating efficiencies, the lack of homogeneity as well as the obscureness of plaques. Trypsin incorporated into the overlay medium rendered chick embryo monolayers as sensitive for Sendai virus plaque formation as monkey kidney monolayers, and this technique could be used for an infectivity assay. This report describes these observations and their application to the infectivity assay of Sendai virus. MATERIALS AND METHODS Virus strains. Three Sendai virus strains, Z, MN and H, were employed. The Z strain, originally isolated from a mouse brain by Okuno and his associates (unpub- 175

H. SHIBUTA, M. AKAMI AND M. MATUMOTO fished data, [5]), was supplied by Dr. N. Yamaguchi, Institute of Medical Science, Tokyo. The MN strain was recovered from a mouse lung by Fukumi et al. [6] and supplied by Dr. K. Tobita, Institute of Public Health, Tokyo. The H strain, isolated from a mouse by Dr. Y. Takeuchi, National Institute of Health, Tokyo (unpublished data) was supplied to us by him. All the strains had been maintained by egg passages. They were grown in the allantoic cavity of 10- day-old chick embryos and infectious allantoic fluids, harvested after incubation at 37 C for 3 days, were used as the seed virus in the present study. Cell cultures. Secondary cultures of cynomolgus monkey kidney cells (MK) were used. Trypsin-dispersed cells, supplied from National Institute of Health, Tokyo, were grown in Roux bottles at 37 C for 7 to 8 days using LE medium (Earle's solution containing 0.5% lactalbumin hydrolysate) supplemented with 3% bovine serum. The cultured cells were dispersed by treatment with PBS(-) (phosphate buffered saline, free of Ca and Mg ions) containing 0.2% trypsin and 0.01% EDTA at room temperature for 15 to 30 min, suspended at a concentration of 2-2.5 ~ 105 cells/ml in Eagle's minimum essential medium (ME) containing 10% tryptose phosphate broth and 5% bovine serum, and seeded in 5 ml amounts in 60 mm petri dishes. The cultures were incubated in a humidified atmosphere of 5% CO2 in air at 37 C for 4 days, when cell monolayers were complete and ready for use. Cultures in tubes (1.5 ~ 15 cm) were prepared by seeding 1 ml of the cell suspension prepared as described above, and incubating in a slanted position at 37 C for 4 days. Chick embryo cells (CE) were used as primary cultures. Ten-day-old chick embryo tissues were trypsinized, and the resulting cells were cultured in petri dishes or tubes as described above for MK cells using 9.0 ~ 106 cells/dish and 1.3 ~ 106 cells/tube. The cultures were ready for use after 1 day of incubation. Primary monolayer cultures of bovine kidney cells (BK) were supplied from National Institute of Animal Health, Tokyo. Those cultures were prepared by the method described previously [17], and incubated at 37 C using YLE medium (LE medium containing 0.1% yeast extract) supplemented with 10% bovine serum. After 7 days of incubation, the cultured cells were used for preparation of secondary cultures as described above for MK cells. All the media employed contained 100 units/ml penicillin and 100 Đg/ml streptomycin. Plaque technique. Monolayer cultures of MK, BK or CE cells prepared in petri dishes, after removal of the culture fluid, were washed once with LE medium, inoculated with 0.1 ml of virus dilution in LE medium, and incubated at 37 C for 60 min. At the end of the incubation, the residual fluid was aspirated and the cultures were covered with 5 ml of agar overlay medium. Various overlay media were used and their composition will be described for each experiment in the text. In some experiments trypsin 1:250 (Difco) was added to the overlay medium at a final concentration of 10 Đg/ml. All the overlay media contained. 0.8% Noble agar, and 100 units/ml penicil- lin plus 100 Đg/ml streptomycin, except medium 199 which contained 60 Đg/ml kanamycin. After incubation at 37 C for a certain period in a humidified CO2 incubator, the cultures received a second agar overlay (4 ml) containing 1:13000 neutral red, and plaques were counted after 24 hr of incubation at 37 C. Tube assay. Tube cultures of MK, BK or CE cells, after removal of the culture fluid, were washed once with LE medium

PLAQUE FORMATION OF SENDAI VIRUS 177 and inoculated with 0.1 ml of tenfold serial virus dilutions made in LE medium. Five tubes were used per dilution. After incubation at 37 C for 60 min, the cultures were fed with 1.0 ml of ME medium containing 5% tryptose phosphate broth and 0.14% bovine serum albumin, and incubated at 37 C for 4 days. At the end of the incubation, the cultures were tested for hemagglutinin by adding 1 ml of 0.5% chicken erythrocyte suspension in PBS(-) solution. Titers were expressed in TCID50 units/ml. Egg assay. Tenfold serial virus dilutions in LE medium were inoculated in 0.1 ml amounts into the allantoic cavity of 10-dayold chick embryos, using 5 eggs per dilution. After incubation at 37 C for 3 days, the allantoic fluid of each egg was tested for hemagglutinin and the EID50 titer was calcula ted. Plaque Formation RESULTS on MK Monolayer In a preliminary experiment, MK monolayers were found to be a sensitive medium for the plaque formation of Sendai virus. We investigated some experimental conditions to develop a plaque count method. The content of sodium bicarbonate in the agar overlay medium affected the plaque formation. Using LE and 199 media, containing 0.14% bovine serum albumin, for agar overlay, various bicarbonate levels of 0.15% to 0.30% were tested, and about 0.2% was found to be adequate for formation of clear plaques. In the following experiments a bicarbonate content of 0.18% was used. Various media, LE, YLE, 199 and ME, for agar overlay were tested for plaque for mation. Those media were supplemented with 0.14% bovine serum albumin. LE medium containing 1% bovine serum was also included in the experiment. Table 1 shows the results with the Z strain. LE, YLE and ME media containing bovine serum albumin and LE medium containing bovine serum produced readily countable, clear plaques. With these overlay media no significant differences in plaque numbers was observed among the media. Medium 199 containing bovine serum albumin, however, tended to be inferior for cell maintenance and the number of plaques produced was somewhat smaller than with the other Table 1. Plaque formation of Z strain on MK monolayers under different agar overlay media media. Table 2. Effects on plaque formation of bovine serum albumin and trypsin added to LE agar overlay medium

178 H. SHIBUTA, MK EZ M. AKAMI AND M. MATUMOTO CE EZ 4d T( + )6d MK EMN CE-MN 4d MK EH 4d T(+)5d CE EH T( +-)6d

PLAQUE FORMATION OF SENDAI VIRUS 179 Further experiments revealed that bovine serum albumin or bovine serum could be omitted from the agar overlay medium without any deleterious effect on the cell maintenance and the plaque size, morphology and number (Table 2). According to these findings we selected LE medium containing 0.8% agar as the overlay medium. Figure 1 shows plaques produced on MK monolayers under this agar overlay with the Z, MN and H strains. The plaques were clearly defined, almost circular, and relatively homogeneous in size. The plaque size varied among the strains. Plaques of the Z strain were 1.5 to 2 mm across, those of the H strain 2 to 3 mm across and those of the MN strain 3 to 4 mm across 4 days postinoculation. With the latter strain it was better to count plaques at 3 days before plaques became too large resulting frequently in fused plaques in crowded dishes having 100 or more plaques. With the Z and H strains the plaque size was too small at 3 days but became adequate for counting at 4 days. Proportionality of Plaque Count to Virus Concentration and Reproducibility in Assay on MK Monolayer By the plaque technique developed in the preceding section the plaque count was shown to be directly proportional to the virus concentration, indicating that infection with one virus particle is sufficient to produce one plaque (Fig. 2). The reproducibility of the titer determined by this assay method was shown to be satisfactory. Table 3 shows results of repeated assays with several different virus stocks stored frozen at -70 C. Fig. 2. Proportionality of plaque count to virus concentration on MK monolayers. Six to 3 dishes were used for each dilution. Table 3. Reproducibility of plaque assay on MK monolayer under LE medium plus 0.8% agar Effect of Try sin in Agar Overlay on Plaque Formation on MK Monolayer Virus stocks of the Z, MN and H strains appropriately diluted in LE medium were inoculated on MK monolayers and the inoculated cultures were covered with LE agar overlay medium containing 10 Đg/ml trypsin. Control cultures received the overlay medium without trypsin. No differences in the size, morphology and number of Fig. 1. Plaques produced by Sendai virus on MK monolayers under overlay of LE medium plus 0.8% agar, and CE monolayers under overlay of medium 199 plus 10 trypsin and 0.8% agar. Left from top: Z, MN and H strains on MK monolayers, 4 (lays postinoculation. Right from top: Z, MN and H strains on CE monolayers, 6, 5 and 6 days postinoculation, respectively.

180 H. SHIBUTA, M. AKAMI AND M. MATUMOTO plaques which were produced was observed between the test cultures and control cultures (Table 2). Plaque Formation on BK Monolayer The Z strain was tested for plaque formation on BK monolayers. We used LE medium (0.18% sodium bicarbonate) containing 0.14% bovine serum albumin and 0.8% agar for overlay. Plaques were produced which were small and non-homogeneous in size and not clearly defined, rendering plaque counting difficult. The situation was not improved after 7 serial passages of the Z strain in BK cultures or 3 consecutive clonings by picking large plaques on BK monolayers, and in addition, plaque titers determined with those viruses on BK monolayers were about 1/5 of those on MK monolayers. Plague Formation on CE Monolayer The plaque formation with the Z, MN and H strains was tested on CE monolayers under agar overlay medium consisting of 199 or LE medium (0.22% sodium bicarbonate) and 0.8% agar, supplemented with 0.14% bovine serum albumin or not. The strains formed, even with relatively heavy inocula, no plaques or very few pin-hole plaques which were discernible with difficulty against the background stained with neutral red. The Z strain, for instance, formed 1 or 2 tiny plaques in dishes received 103 PFU of virus as determined by the MK plaque count method. Cloning from these plaques failed to show an enhanced plaque-forming capacity of the virus. Enhanced Plaque Formation on CE Monolayer by Try sin Incorporated in Overlay Medium However, when trypsin was incorporated into the overlay medium at a concentration of 10 Đg/ml, clear plaques were obtained. The addition of bovine serum albumin to the trypsin-containing overlay medium rendered plaques less clearly defined. The use of medium 199 for agar overlay gave better results than that of LE medium. Plaques formed under the former agar overlay were large enough to be readily counted, while those with the LE overlay were smaller, particularly with the Z strain which formed pin-hole to 0.5 mm plaques 5 days after inoculation. Under the 199 overlay, plaques with the MN and H strains became readily countable, 2 to 3 mm and 1 to 1.5 mm across respectively, 5 days postinoculation, but those with the Z strain were still small, 0.5 to 1 mm across, 5 days postinoculation and became countable, 1 to 1.5 mm across, 6 days postinoculation. Figure 1 shows plaques formed with the three strains on CE monolayers under agar overlay consisting of medium 199 and 0.8% agar. This technique gave infectivity titers comparable to those obtained on MK monolayers (Table 4). Table 4. Parallel titrations by plaque assay on MK and CE monolayers Parallel Titrations by Egg, Tube and Plaque Assay Methods Stock No. 12 of the Z strain was titrated by the allantoic cavity inoculation of 10-

PLAQUE FORMATION OF SENDAI VIRUS 181 day-old chick embryos, and the tube and plaque assay methods using MK, BK and CE cell cultures. The egg inoculation and the tube assay methods were performed as described in Materials and Methods. The plaque assay was carried out using, as the overlay medium, LE medium plus 0.8% agar for MK, LE medium plus 0.14% bovine serum albumin and 0.8% agar for BK, and medium 199 plus 10 Đg/ml trypsin and 0.8% agar for CE. As summarized in Table 5, the MK and CE PFU titers and the MK TCID50 titer were similar to the titer. The BK TCID50 and PFU titers were about one-log unit lower than the above titers. The CE TCID50 titer was markedly low. Table 5. Parallel titrations of No. 12 stock of Z strain by various assay methods : egg inoculation, and tube and plaque methods using MK, BK and CE cell cultures DISCUSSION The present data indicate that cynomolgus monkey kidney cell monolayers are a highly sensitive medium for plaque formation of Sendai virus, and provide a practical method for the infectivity assay of the virus. This method gave reproducible results, and was as sensitive as the egg inoculation or the tube culture method using monkey kidney cells, and simple enough to be used routinely. With some other members of the parainfluenza virus group, plaque formation on monkey kidney cell monolayers has also been reported, and the method has shown utility in the assay of virus infectivity. Thus, Tytell et al. [18] described a method for plaque formation of parainfluenza virus type 2 in grivet monkey kidney cells. Choppin [2] reported that SV5 virus, a simian parainfluenza virus, multiplied to high titers in rhesus monkey kidney cells, and formed plaques with high efficiency. Another finding of interest in the present study is the enhancement of plaque formation by Sendai virus on chick embryo cell monolayers by the incorporation of trypsin into the overlay medium. Similar observation using pancreatin was made by T. Matsumoto and his associates (personal communication). Under the appropriate conditions determined in the present study, the plaque count obtained by this technique was comparable to that on monkey monolayers. This is a great contrast to the fact that virtually no recognizable plaques were formed under the overlay medium without trypsin. Colobert and Louisot [3] reported that KB cells infected with Sendai virus, which produced no recognizable cytopathic effect, developed cytopathogenesis and produced high-titered active virus, when the cells were treated with trypsin 5 or 6 days post-

182 H. SHIBUTA, M. AKAMI AND M. MATUMOTO infection. Enhanced replication of parainfluenza virus type 4 by trypsin, pancreatin and pronase in Vero cells, established line cells of African green monkey kidney origin, was reported by Morimoto et al. [10]. Similar enhancing effect of trypsin on replication of parainfluenza virus type 3 in HEp- 2 cells [13] and HA2 virus of parainfluenza virus type 1 in Vero cells [12] was reported. Such an enhancement of viral replication would probably be shown in our case, but this is to be confirmed. Gifford and Klapper [8], however, showed that the final yield of vaccinia virus at the end of one cycle of replication was the same in enzyme-treated. and control cultures, but the virus multiplication was speeded up by about 5 hr in treated cultures. Enhanced plaque formation by proteolytic enzymes has been reported for reoviruses by chymotrypsin, trypsin and papain [15, 16], and pancreatin [4, 19], vaccinia virus by trypsin and chymotrypsin [7, 8], enteroviruses by pancreatin [20], influenza A and B viruses by pancreatin and trypsin [1], and parainfluenza virus type 3 [13] and type 4 [10] by trypsin. Spendlove and Schaffer [16] suggested that the enhanced plaque formation of reoviruses by proteolytic enzymes resulted from the action of the enzyme on a virus-associated substrate. Lerner et al. [9], however, reported that trypsin treatment of reovirus type 2 or Coxsackie B5 had little or no effect on infectivity, but similar treatment of reovirus type 1 increased infectivity titers. According to Gifford and Klapper [8], Spendlove found more recently that the enhancement of reovirus infectivity was due to the removal of the outer viral capsid. On the other hand, Came et al. [1] suggested that the enhancement of influenza virus plaque formation by pancreatin resulted from the action of the enzyme on the cells rather than directly on virus particles or on a component of the overlay medium. According to Gifford and Klapper [8], the enhancement of plaque number of vaccinia virus was due to an effect of the enzyme on the virus and the enhancement of plaque size was due to an effect on the cells. There is also the possibility that the proteolytic enzyme destroys interferon produced by infected cells and thereby allows virus form plaques. These possibilities suggested by the previous workers should also be investigated for the elucidation of the mechanism of the phenomenon in our virus-host system. Nagata et al. [11] reported that their plaque count method using calf kidney cells gave titers similar to E1D50 titers. Our data showed lower titers by plaque counting in calf kidney cells than by the egg inoculation method. However, a difficulty in using calf kidney cells arose from the fact that plaques formed by our strains were small and non-homogeneous in size and were not clearly defined. Non-homogeneity in the plaque size was also described by Nagata et al. [11]. This situation would, of course, affect the reliability of plaque count. We have no experience using the plaque count in kidney cells of newly hatched chicks described by Shigeta [14]. This method seemed less sensitive than the plaque assay on monkey kidney monolayers since the plaque titer on chick cells was reported to be about 1/3 to 1/10 of the EID50 titer [14]. These considerations seem to indicate that the plaque assay on monkey kidney monolayer is the method of choice for the infectivity titration of Sendai virus. The use of chick embryo cells with an overlay medium containing trypsin may also be used, although further data is needed for its standardization.

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