Rhinovirus Plaque Formation in WI-38 Cells with

APPLIE MICRoBIoLoGY, Sept. 1968, p. 1331-1336 Copyright @ 1968 American Society for Microbiology Vol. 16, No. 9 Printed in U.S.A. Rhinovirus Plaque Formation in WI-38 Cells with Methylcellulose Overlay TIMOTHY M. DOLAN,' JAMES D. FENTERS,2 PATRICIA A. FORDYCE,3 AND JACOB C. HOLPER Devartment of Infectious Diseases, Scientific Division, Abbott Laboratories, North Chicago, Illinois 60064 Received for publication 13 June 1968 A sensitive, reliable plaque assay system is described for five rhinoviruses using freshly prepared methylcellulose overlay and human embryonic diploid cells. Circular plaques with irregular edges, 2 mm in size, were formed by rhinoviruses 1A, 2, 6, and 13 after 6 or 7 days of incubation. A fifth rhinovirus, 17, formed a 1- to 2-mm feather plaque after 14 days of incubation. Plaque counts of rhinoviruses 1A and 13 were not affected by varying the ph of the overlay from 6.9 to 7.5. Plaque sizes and plaque-forming unit values of high passage rhinoviruses IA and 13 were equivalent when tested at 26, 31, or 36 C. The rhinoviruses tested were sensitive to incubation at 40 C or heating at 50 C. Enhancement of plaques was observed when Mg++ was incorporated into agar overlays, but enhancement did not occur when Mg++ was added to methylcellulose overlays. The plaque assay for animal viruses was first described by Dulbecco (2) and has since found widespread application to many virus-cell systems, incluiding rhinoviruses (5, 11, 13, 14, 18). The original method of observing small foci of degenerating cells infected by rhinoviruses under the microscope (13) was replaced by the production of plaques under agar (14). Plaque characteristics such as shape, size, and degree of lysis of cells caused by different rhinoviruses may be observed under agar (5, 11, 14, 18). Variations in the use of primary tissues or human heteroploid cells, methylcellulose or agar overlay, or additives such as diethylaminoethyl (DEAE)-detran or Mg++ to the overlay have been described (5). Genetic markers such as the bicarbonate (d) marker (16) or temperature (T) markers (12) may be performed with a plaque assay system. Methylcellulose is an agar substitute first described by Hotchin (7) in plaque assays of herpes simple virus. The advantages of it are that it is nontoic (7), chemically defined (15), avoids inhibitory effect of agar (15), and it is relatively easy to handle (7). The formation of plaques with rhinovirus 2 in heteroploid cells using methylcellulose has been reported (5). The purpose of this paper is to present a sensitive, reliable assay system for five 1 Present address: University of North Dakota, Department of Microbiology, Grand Forks, N.D. 21Present address: Life Sciences, IIT Research Institute, Chicago, Ill. 60616. 3Present address: G. D. Searle, P.O. Bo 5110, Chicago, Ill. 60680. rhinoviruses using the methylcellulose system with human diploid cells. assay MATERIALS ANDW METHODS Tissue culture. The human embryonic diploid cell strain, WI-38, used in these eperiments was originally obtained from Flow Laboratories, Inc., Rockville, Md. and from L. Hayflick of the Wistar Institute Philadelphia, Pa. The methods used for the passage of WI-38 cells were as described by Hayflick and Moorhead (6). Growth medium was Eagle's basal essential medium (BME; Flow Laboratories, Inc.) with the addition of 10% fetal bovine serum, 50 units per ml of polymyin, and 100lg per ml of neomycin sulfate. Confluent monolayers of WI-38 cells were obtained in flasks (Falcon Plastics, Los Angeles, Calif.). Overlays. A 3% stock methylcellulose (400 centipoises, Fisher Chemical Co., Pittsburgh, Pa.) solution was prepared with triple distilled water and autoclaved at 124 C for 20 min. As the methylcellulose cooled to room temperature, it was occasionally rotated by hand to effect the solution. Immediately after the methyl-cellulose dissolved, an equal volume of twofold concentration of media with 10% fetal calf serum (Hyland Laboratories, Los Angeles, Calif.) was added. The concentrated media consisted of double volumes of vitamins, L-glutamines, and essential amino acids in a 2X solution of Earle's BSS (2X BME). Modified lactalbumin hydrolysate (14), modified Earle's BSS (10), or a 2X solution of 199 were used in some eperiments. Final concentrations of 1.25% Noble agar or Difco agar were used in combination with different media. DEAE-detran (Pharmacia, Uppsala, Sweden) or 30 mm MgC12 was incorporated into some eperiments to note if plaque size enhancement occurred. Plaque assays. Monolayers of WI-38 cells were used 1331

1332 DOLAN ET AL. APPL. MICROBIOL. 4 to 6 days after seeding the flasks. Prior to virus inoculation, the cell sheet was washed once with 2 ml of Earle's BSS, the supernatant fluid was decanted, and 2 ml of Earle's BSS was added. An 0.1-ml amount of the appropriate dilution of virus was added to each of three flasks, with absorption for 2 hr at room temperature. The overlays were added to the flasks in 5-mi volumes using a Cornwall automatic syringe. At the end of the 6- to 7-day incubation period at 33 C, the methylcellulose overlay was decanted. A 4-nd amount of absolute methanol was added to each flask for 10 min. The alcohol was removed, and 4 ml of a 1:10 stock solution of Giemsa stain (Difco) was added for 30 min. In eperiments using agar, a second overlay containing a 1:20,000 final concentration of neutral red was added, and the flasks were incubated for 4 hr at 36 C. Linearity and temperature eperiments. Linearity eperiments were performed according to the method of Dulbecco and Vogt (3) using twofold dilutions of virus. The temperature genetic markers, T26, T30, and T40, were determined by incubating the test for 7 days at the stated temperature (12). The T50 marker was determined by the method of Hozinski et al. (8), ecept that the viruses were heated for 30 min at 50 C in our system before plaque titrations. Viruses. Rhinoviruses 1A, 2, 6, 13, and 17 were used. The history of the viruses was given in an earlier publication (4). The nomenclature used is as approved by the Directors of the World Health Organization Respiratory and Enterovirus Reference Centers (9). Rhinoviruses 1 and 13, which had been passed twice in WI cells, were kindly supplied by Vernon Knight, College of Medicine, Baylor University, Houston, Te. Neutralization tests. An 0.3-nl amount of a 1:20 dilution of heat-inactivated (56C, 30 min) bovine antisera (Abbott Laboratories, North Chicago, Ill.) was added to 0.3 ml of twofold dilutions of viruses, incubated at room temperature for 2 hr. An 0.1-ml amount of this miture was inoculated into each of three flasks containing WI-38 cells. Absorption was for 2 hr at room temperature. After decanting the fluids, the BME-methylcellulose overlay was added, and the test was incubated for 7 days at 33 C. The cells were then stained with Giemsa stain, and the plaques were counted. RESULTS Determination of overlay for rhinovirus plaques. Rhinoviruses 1A, 2, 13, and 17 produced consistent macroplaques with BME and methylcellulose on WI-38 cells (Table 1). The use of modified lactalbumin hydrolysate or modified 199 with methylcellulose led to the formation of microplaques, ecept with rhinovirus 2 which formed macroplaques- with modified 199 and methylcellulose. By contrast, under agar, only microplaques or faint hazy macroplaques were produced by four rhinoviruses. Due to the lack of a strong background stain with neutral red, the plaques were difficult to observe, and the TABLE 1. Overlay' LH, FCS, NA.. LH, FCS, MC.. 199, FCS, NA.. 199, FCS, MC.. BME, FCS, NA BME, FCS, NA, D... BME, FCS, DA, D... BME, FCS, MC... BME, CS, MC.. Earle's, CS, DA, D... Selection of overlay for rhinovirus plaques I~~~~~~~~~~~~~ Rhino- Rhino- Rhino- Rhinovirus virus virus virus IA 2 6 13 4b 0 + + -+- Rbinovirus 17 a"lh, modified lactalbumin hydrolysate; FCS, fetal calf serum; NA, Noble agar; MC, methylcellulose; BME, basal medium, Eagle; D, DEAEdetran; DA, Difco agar; CS, agamma chicken serum. b Symbols: +, macroplaques; i, microplaques; 0, negative;-, not tested; X, faint hazy macroplaques. method was discarded in favor of the methylcellulose overlay. Optimal number of plaques of rhinoviruses were produced at 31 or 33 C using methylcellulose, BME, and either agammaglobulin chicken serum or fetal calf serum (Table 2). Using fetal calf serum in the overlay, equivalent plaqueforming unit (PFU) values were obtained at 31, 33, and 35 C with rhinoviruses IA and 13. With the same overlay, the PFU values of rhinovirus 2 appeared to decrease with increasing temperature. The PFU values of rhinoviruses la and 13 also decreased with increased temperature with the agammaglobulin chicken serum overlay. The PFU to TCID50 ratio was approimately 1:1 at 33 C but ranged from 1:2 to 1:20 at 35 C with rhinoviruses 1A and 13, respectively, when agamma chicken serum was used. Some granulation was observed in the monolayers containing chicken serum which was not observed with fetal bovine serum. Virus stock of either rhinovirus la or 13 did not vary more than twofold when titrated on different passages of WI-38 cells over a 6-month period of time (Table 3). Figures 1, 2, and 3 demonstrate the number of macroplaques formed and their size with rhinoviruses 1A, 2, and 13. Maimal plaque titer was obtained in 4 to 5 days with the three rhinoviruses at 33 C using BME and methylcellulose. +

VOL. 16, 1968 RHINOVIRUS PLAQUE FORMATION IN WI-38 CELLS 1333 TABLE 2. Effect of temperature and serum on rhinovirus plaques using methylcellulose overlay Ept Type serum in 31 C 33 C 35 C PU Size PU Size H F Size PU (MM) PU (MM) (mm)u Rhinovirus 1A I Chicken 4.5 X 102-2.5 X 102-1O25 5.0 X 101-102.0 II Fetal calf 1. 3 X 104 2 Not tested - - 1.4 X 104 - Rhinovirus 2 I Fetal calf 8.4 X 104 2 1.3 X 104 2-4.0 X 103 1.5 II Fetal calf Not tested - 4.2 X 108 2-4.0 X 102 1 Rhinovirus 13 I Chicken 6.3 X 102-4.5 X 102-102.5 5.0 X 101-103- II Fetal calf 9.4 X 102 2-5 1.3 X 104 2-5 - 2.0 X 104 3-7 III Fetal calf 1.4 X 104 3 Not tested - - 1.2 X 104 2 - TABLE 3. Rhinovirus plaque formation on different WI-38 passages Virus Ept no. PFUFrml Rhinovirus 1A, passage I 3.3 X 104 no. 6 II 5.9 X 104 III 6.0 X 104 IV 3.6 X 104 V 6.5X 104 Rhinovirus 13, passage I 2.0 X 104 no. 13 II 4.0 X 104 III 2.2 X 104 Rhinovirus 13, passage I 1.2 X 104 no. 15 II 1.3 X 104 Microscopic plaques formed in 3 days and increased to 1 mm in 4 days. The plaque size of rhinoviruses 1A and 13 continued to increase with time (Fig. 1 and 3). Rhinoviruses IA and 13 increased to 5 to 6 mm by 10 days, whereas the plaque size of rhinovirus 2 remained relatively constant. Plaques of approimately a 2-mm size were read after 6 or 7 days of incubation. A gradual fading of plaques was observed over a 21-day period due to increasing plaque size and viability of the WI-38 cells. The three rhinoviruses tested and also rhinovirus 6 formed a circular plaque with irregular edges. Rhinovirus 17 formed a 1- to 2-mm feather plaque in 14 days. The plaque formed could be classified as turbid because of incomplete lysis of the cells. The plaque assay system using methylcellulose with rhinoviruses 1A, 2, and 13 showed a linear relationship when twofold dilutions of virus inoculum were plotted against the number of PFU formed. Neutralization tests using a 1:20 dilution of bovine antisera against the various samples gave complete suppression of plaque formation in all dilutions tested. Effect ofph on plaque formation. Rhinoviruses 1A and 13 were not affected by the ph of the overlay (Table 4). A decrease in plaque size but not count was noted for both viruses at ph 7.5. A slight decrease in plaque size occurred with rhinovirus 1A at ph 6.9. Effect of Mg on plaque formation. Eperiments were conducted to determine if enhancement of plaque size occurred when 30 mm MgC12 was added to either Difco agar or methylcellulose overlays (Table 5). Significantly higher PFU values were obtained when Mg++ was present in the agar overlay. The plaque size of rhinovirus 13 was increased by 2 to 3 mm in the presence of 30 mm MgC92. Slightly increased titers (two- to fourfold) were noted when Mg++ was present in the methylcellulose overlay, but plaque size enhancement did not occur. The titers of the two rhinoviruses were similar when methylcellulose without Mg+ and agar with Mg+ were compared. Effect of tempertaure on plaque formation. Preliminary eperiments with rhinovirus IA at two different passage levels (WI-2 and WI-6) indicated plaques formed equally well at 26, 31, and 36 C with few or no plaques being formed at 40 and 50 C (Table 6). A high passage (WI-I 3) isolate of rhinovirus 13 had similar PFU values and sizes at 26, 31, and 36 C with no plaques observed at 40 or 50 C. A low passage (WI-2) isolate of rhinovirus 13 formed delayed plaques at 26 C. The values of the low passage isolate were equivalent at 31 and 36 C, whereas an additional 2 to 3 days of incubation at 26 C was required for the plaques to become microscopically visible. All rhinoviruses tested, ecept the low passage isolate of rhinovirus 13 at 26 C, had a plaque size of approimately 2 mm when counted at 6 to 7 days. DIscussIoN Plaque assays for five rhinoviruses were performed with human embryonic diploid cells

1334 DOLAN ET AL. APPL. MICROBIOL. FIG. 1. Rhinovirus IA plaque formation at different days. From left to right, bottles represent days 2, 3, 4, 6, 7, 9, 10, 21. FIG. 2. Rhinovirus 2 plaque formation at different days. From left to right, bottles represent days 2, 3, 4, 6, 7, 9, 10, 21. FIG. 3. Rhinovirus 13 plaque formation at different days. From left to right, bottles represent days 2, 3, 4, 6, 7, 9, 10, 21. (WI-38) and a simple overlay medium consisting of BME, methylcellulose, and fetal bovine serum. The critical factor for plaque production appeared to be the preparation of methylcellulose. Fiala and Kenny (5) found that rhinovirus 2 produced a plaque in calf HeLa cells of only 0.5 mm with methylcellulose, whereas we observed that 2-mm plaques were produced in the human diploid

VOL. 16, 1968 RHINOVIRUS PLAQUE FORMATION IN WI-38 CELLS TABLE 4. Effect of ph on1 rhinovirus plaques 1 335 TABLE 5. Effect of MgCl2 otn rhinovirus plaques with agar anid methylcellulose overlaysa Agar Agar Mgd' Methylcellulose Methylcellulose Mg++ Virus VirusEpt'FU Size Size Size Size PFU (mm) PU (mm) PU (mm) PU (m Rhinovirus 2 I 1.6 X 102 0.5-1 3.4 X 104 0.5-1 TNTCb 0.5-1 1.3 X 104 0.5-1 Il 4.5 X 102 0.5-1 9 X 103 0.5-1 3.0 X 104 1 8.0 X 104 1-2 Rhinovirus 13 1 2.9 X 103 1-3 2.5 X 104 3-5 2.8 X 103 1 1.1 X 104 1 _ 1.2 X 104 2 5.0 X 104 2-5 3.7 X 104 1-5 7.4 X 104 1-4 a Methylcellulose stored at 4 C. Too numerous to count. TABLE 6. Temperature (T) markers of rhin7oviruses Virus Ept T26 T26 delayeda T31 T36 T40 T50 Rhinovirus la, P2... I 7.0 X 104 None 1.2 X 105 4.6 X 104 <102.0 1.0 X 102.0 Rhinovirus1A, P6... I 1.3 X 104 None 1.7 X 104 1.9 X 104 < 1010 <101.0 Rhinovirus 13, P2... I <102 Not tested 9.9 X 103 2.5 X 104 <102.0 <102.0 Rhinovirus 13, P13... I 4.5 X 103 None 1.4 X 104 1.2 X 104 <1010 <1010. Rhinovirus 13, P2... II <102 2.2 X 102 2.0 X 103 7.0 X 102 1.0 X 102 2.0 X 102 Rhinovirus 13, P13... II 1.1 X 104 None 2.2 X 104 8.0 X 102 <101.0 <101.0 a Small plaques form after 9 days of incubation instead of the usual 6 to 7 days. system when the methylcellulose overlay was prepared as directed. Eperiments in which methylcellulose was stored at 4C prior to use indicated that smaller plaques were formed with rhinoviruses IA and 13 than when freshly prepared methylcellulose was used. When the methylcellulose remained at room temperature for 2 hr after being in solution, or when the methylcellulose was chilled at 4 C after autoclaving, the viscosity changed so that the size of the plaques formed was inhibited. A vibration-free incubator, such as a room maintained at the desired temperature, was essential to prevent the formation of poorly defined plaques or secondary plaques. The optimal temperature for size and number of plaques formed ranged from 31 to 36 C for rhinoviruses IA and 13, but plaque production with rhinovirus 2 diminished significantly at 36 C. Rhinovirus 2 was earlier reported to be inhibited at 36 C (13). Cate et al. (1) described a model system using rhinovirus 14 for testing survival and replication of rhinoviruses in the intestinal tract of human volunteers and found its growth inhibited in WI-38 cells at 37 C, thus suggesting a possible mechanism of failure for the rhinoviruses to infect the intestine or stimulate antibody production. With the implication that other rhinoviruses (1A and 13) do multiply at 36 C, further studies should probably be performed on different prototypes. The significance of the delayed 26 C marker with a low passage rhinovirus 13 has not been determined. Circular, irregular edged turbid plaques were formed after 6 to 7 days of incubation at 33 C with rhinoviruses 1A, 2, 6, and 13 (2 mm in size). A fifth rhinovirus, 17, formed a 1-mm feather plaque after 14 days of incubation. A ph range of the overlay from 6.9 to 7.5 did not affect the plaque count, although plaque size decreased at the more alkaline ph. The addition of 30 mm MgCl, in agar overlays enhanced both the PFU value and the size of the rhinoviruses tested as reported by Fiala and Kenny (5). They also observed that doses up to 40 mm Mg++ increased

1336 DOLAN ET AL. APPL. MICROBIOL. rhinovirus 2 plaque size when using methylcellulose overlay and a sensitive human heteroploid cell line. In our studies with the human diploid cell strain WI-38, the incorporation of Mg++ into the methylcellulose overlay did not enhance titers or plaque sizes significantly. This suggests that the Mg++ ion has its effect with the agar or agar-cell system as reported by Walfis et al. (17) with enteroviruses. ACKNOWLEDGMENT This investigation was supported by the Vaccine Development Board of the National Institutes of Health under contract PH 43-62-487. LITERATURE CITED 1. Cate, T. R., R. G. Douglas, Jr., K. M. Johnson, R. B. Couch, and V. Knight. 1967. Studies on the inability of rhinovirus to survive and replicate in the intestinal tract of volunteers. Proc. Soc. Eptl. Biol. Med. 124:1290-1295. 2. Dulbecco, R. 1952. Production of plaques in monolayer tissue cultures by single particles of an animal virus. Proc. Natl. Acad. Sci. U.S. 38:747-752. 3. Dulbecco, R., and M. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Eptl. Med. 99:167-182. 4. Fenters, J. D., S. S. Gillum, J. C. Holper, and G. S. Marquis. 1966. Serotypic relationships among rhinoviruses. Am. J. Epidemiol. 84:10-20. 5. Fiala, M., and G. E. Kenny. 1966. Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium. J. Bacteriol. 92:1710-1715. 6. Hayflick, L., and P. S. Moorhead. 1961. The serial cultivation of human diploid cell strains. Eptl. Cell Res. 25:585-621. 7. Hotchin, J. E. 1955. Use of methylcellulose gel as a substitute for agar in tissue culture overlays. Nature 175:352. 8. Hozinski, V. I., V. B. Seibil, N. S. Pantelyeva, S. M. Mazurova, and E. A. Novikova. 1966. The rct4o and T50 markers and characteristics of some variants of measles virus. Acta Virol. (Prague) 10:20-27. 9. Kapikian, A. Z. (Chairman), et al. 1967. Rhinoviruses: a numbering system. Nature 213:761-763. 10. Kisch, A. L., and K. M. Johnson. 1963. A plaque assay for respiratory syncytial virus. Proc. Soc. Eptl. Biol. Med. 112:583-589. 11. Kisch, A. L., P. A. Webb, and K. M. Johnson. 1964. Further properties of five newly recognized picomaviruses (rhinoviruses). Am. J. Hyg. 79:125-133. 12. Lwoff, A., and M. Lwoff. 1958. L' inhibition du developpement du virus poliomyelitique a 390 et le probleme du role de l'hyperthermie dans l'evolution des infections virales. Compt. Rend. 246:190-192. 13. Parsons, R., and D. A. J. Tyrrell. 1961. A plaque method for assaying some viruses isolated from common colds. Nature 189:640-642. 14. Porterfield, J. S. 1962. Titration of some common cold viruses (rhinoviruses) and their antisera by a plaque method. Nature 194:1044-1047. 15. Schulze, I. T., and R. W. Schlesinger. 1963. Plaque assay of dengue and other group B arthropod-borne viruses under methylcellulose overlay media. Virology 19:40-48. 16. Vogt, M., R. Dulbecco, and H. A. Wenner. 1957. Mutants of poliomyelitis viruses with reduced efficiency of plating in acid medium and reduced neuropathogenicity. Virology 4:141-155. 17. Wallis, C., J. L. Melnick, and M. Bianchi. 1962. Factors influencing enterovirus and reovirus growth and plaque formation. Teas Rept. Biol. Med. 20:693-702. 18. Webb, P. A., K. M. Johnson, and M. A. Mufson. 1964. A description of two newly-recognized rhinoviruses of human origin. Proc. Soc. Eptl. Biol. Med. 116:845-852.