Studies on Cytotropism in Animal Viruses

JOURNAL OF BACTERIOLOGY, Oct., 1966 Copyright 1966 American Society for Microbiology Vol. 92, No. 4 Printed In US.A. Studies on Cytotropism in Animal Viruses III. Growth of Influenza Virus in Epithelial-like and Fibroblastic Cells Derived from Chick Embryo Lung ROBERT B. STEWART' AND PAUL H. FRICKEY2 Department of Microbiology, School of Medicine and Dentistry, University oj Rochester, Rochester, New York Received for publication 16 June 1966 ABSTRACT STEWART, ROBERT B. (University of Rochester, Rochester, N.Y.), AND PAuL H. FRICKEY. Studies on cytotropism in animal viruses. III. Growth of influenza virus in epithelial-like and fibroblastic cells derived from chick embryo lung. J. Bacteriol. 92:972-977. 1966.-Growth of the PR8 strain of influenza virus was studied in epithelial-like and fibroblastic cell cultures derived from chick embryo lungs. The cells were found to differ in morphology, staining characteristics, and in their ability to support production of infectious influenza virus. Fibroblastic cells were characterized by their spindle shape, content of a mucopolysaccharide, their relative inability to synthesize infectious influenza virus, and production of a cell-associated noninfectious hemagglutinin. Epithelial-like cells were characterized by their polygonal shape, absence of mucopolysaccharide, and ability to synthesize infectious influenza virus. In a previous study (16), it was found that cell cultures derived from chick embryo lungs were able to produce higher levels of infectious influenza virus than were similar cultures obtained from lungs of hatched chicks. It was noted that islands of epithelial-like cells were numerous in cultures from embryo lungs, whereas fibroblastic cells characterized the cultures prepared from lungs of hatched chicks. It was also found that cultures composed principally of epithelial-like cells gave rise to significantly higher titers of infectious influenza virus than did cultures in which fibroblastic cells predominated. Furthermore, no differences were noted in the virussynthesizing capacity between epithelial cell cultures from chick embryos or hatched chicks. The present study was carried out to characterize more fully the differences in two cell types derived from the same organ with respect to their capacity to synthesize influenza virus. Tissue cultures. Sacrifice of 18-day chick embryos was accomplished by decapitation, and the lungs were aseptically removed and washed with Hanks (7) balanced salt solution (BSS) containing 1 units of penicillin and streptomycin per milliliter. The lungs were minced with cataract, knives, and the minced tissue was treatedwitho.1% Trypsin (1:25; Difco) diluted in Hanks BSS by use of a magnetic stirrer. After 1 min of trypsin treatment at 37 C, the larger tissue fragments in the suspension were allowed to settle for 3 min. The supernatant fluid, containing mainly single cells, was centrifuged, and the cells were collected and suspended in Puck's (12) F4 medium, containing 1% fetal calf serum (F4FCS), to give a concentration of 8 X 1' to 9 X 1 cells per milliliter. This suspension was added to sterile tubes and incubated at 37 C in a CO2-gassed incubator for 4 to 5 days with daily changes of the medium. At this time, monolayers of fibroblastic-like cells had formed. The larger fragments of lung tissue which had settled during the first treatment with trypsin were washed with Puck's (11) saline A and trypsin-treated as before for 1 hr at 37 C. At the end of this time, the MATERIALS AND METHODS material was filtered through stainless-steel mesh Virus. The PR8 strain of influenza type A virus was (mesh size, 15), and the cells and small fragments the same as used previously (16). Infective allantoic were collected by centrifugation. This material was fluids stored at -6 C were thawed and diluted in then suspended in Hanks BSS containing.5% lactalbumin hydrolysate,.1% yeast extract, and 1% fetal Puck's (12) saline F for the infection of cells. calf serum, to give a cell count of 18 cells per milliliter. 1 Present address: Department of Microbiology and The medium used in the next two daily changes was Immunology, Queen's University, Kingston, Ontario, F4FCS containing 1 mg/1 ml of prednisolone Canada. sodium succinate (Schering Corp., Bloomfield, N.J.), 2 Present address: Lederle Laboratories, Pearl which was added to suppress growth of fibroblastic River, N.Y. cells (5). Thereafter, the same medium was used with- 972 Downloaded from http://jb.asm.org/ on January 14, 219 by guest

VOL. 92, 1966 CYTOTROPISM IN ANIMAL VIRUSES 973 out prednisolone. A monolayer of epithelial-like cells usually formed after 4 days of incubation at 37 C in a CO2 incubator. Receptor-destroying enzyme (RDE). RDE (Behringwerke, Marburg, Germany) was diluted in saline F to give about 12 units per 1.5 ml. Virus assay. Infectious virus was assayed in embryonated hens' eggs as described previously (16), and the results were expressed at logoeid5o/ml. When RDE was present in the material to be titrated, the dilutions were made in phosphate-buffered saline containing 1% sodium citrate. Hemagglutination titrations were carried out by use of twofold serial dilutions in phosphate-buffered saline, and by adding an equal volume (.5 ml) of chicken red blood cells, (RBC,.5%). The hemagglutinin titer, in units per milliliter, was expressed as the reciprocal of the highest dilution showing positive hemagglutination. Hemadsorption. The hemadsorption technique used was a modification of that of Shelokov et al. (14). After infection of either cell type with input multiplicities of 1 to 12 Eu5 per cell, attachment of virus was allowed to proceed for 2 hr at 4 C. Cultures were then washed five times with saline F, and 12 units of RDE was added to each culture. After 3 min of incubation at. 37 C, which was sufficient to remove superficially attached virus, the cultures were washed as before, and F4FCS medium was added. At various time intervals during incubation at 37 C, media were removed from cultures, and the cultures were then washed three times with saline F before the addition of 1. ml of a 5% suspension of chicken RBC. After allowing sufficient time for hemadsorption to take place, the RBC suspension was removed, and the cultures were gently washed with saline F. Of the 6 to 1, cells counted, the percentage demonstrating hemadsorption was determined. Histochemical staining. Cover-slip preparations of cells were stained by the May-Grunwald Giemsa method (1) and by the Rinehart and Abul-Haj (13) method for acid mucopolysaccharides to characterize differences in the two cell types. LUJ x REsULTS Tissue culture system. The epithelioid cells obtained in culture by the use of steroid and differential trypsin treatment could be distinguished from the fibroblastic cells on the basis of morphology, content of mucopolysaccharide, and susceptibility to infection with the PR8 strain of influenza virus. Cover-slip preparations of both cell types stained by the May-Grunwald Giemsa method showed the epithelioid cells to be polygonal in shape, in contrast to the spindle shape of the fibroblastic cells which tended to be oriented in parallel lines. The colloidal iron staining method showed the same morphological distinctions, but, in addition, the fibroblastic cells stained blue, indicating the presence of mucopolysaccharide. This material could not be identified as hyaluronic acid by the use of hyaluronidase (75 turo * ST-. PASG ULUE*,j 2 -PRIMARY CULTURE * ** I IST. PASSAGE CULTURE*-.. 6TH PASSAGE CULTURE... STANDAR D DE VIATION I. O 2S 4 6 8 1 12 14 FIG. 1. Growth of influenza virus in epithelial-like cells carried through several passages. w 6 PRIMARY CULTURE 1ST PASSAGE CULTURE 5 \ 6TH. PASSAGE CULTURE 1 STANDARD DEVIATION I x \ 45% f....12 -i O \.-... 2 4 6 8 1 12 14 FIG. 2. Growth of influenza virus in fibroblastic cells carried through several passages. bidity-reducing units), because the blue staining material was not removed by the enzyme. This result does not necessarily preclude the possibility that hyaluronic acid is the material present, because Erichsen et al. (3) were also unable to remove an intracellular mucopolysaccharide with hyaluronidase and raised the question of permeability of the fixed cells to the enzyme. The epithelial-like cells, in contrast, showed no blue staining and stained the pink of the counterstain. These results are in agreement with those of Grossfeld (6), who also was able to distinguish epithelial from fibroblast cells on the basis of mucopolysaccharide content. Growth ofinfluenza virus in primary and passage lines of epithelioid and fibroblastic cells. Cultures of both cell types were prepared as previously described, and, when monolayers had formed in ';. Downloaded from http://jb.asm.org/ on January 14, 219 by guest

974 STEWART AND FRICKEY J. BACTERIOL. the culture tubes, the growth medium (F4FCS) was removed and the cultures were washed thoroughly with saline F. The virus inoculum was added in small volumes (.2 ml) in cold saline F to give a multiplicity of between.1 and.3 EID5 per cell. After 2.5 hr of attachment at 4 C, the cells were washed to remove the majority of unattached virus, and growth medium was again added. The tissue culture fluids were completely removed for virus assay every 24 hr, and replaced with fresh medium. The results of these experiments are shown in Fig. 1 and 2, where each point on the graphs represents the mean titer of three samples and each sample is the pool of fluids from two cultures. Figure 1 shows that primary, first-passage, and sixth-passage cultures of epithelial-like cells supported good levels of influenza virus growth, the maximal yields being 1 to 1, times greater than the input virus. Further evidence of the virus-synthesizing efficiency of the epithelioid cells was the finding that the ratios of infectious virus to hemagglutinin (logi EID5o/HA) were of the order of 4. to 5.6. The lower values of the ratio of infectious virus to hemagglutinin were found in the sixth-passage cultures. The only explanation as to why a greater proportion of presumably noninfectious hemagglutinin was produced in these cultures was the finding that the 1lglo EID5o/HA ratio of the inoculum was lower (4.5) than that used with primary and first-passage cultures (5.5). The results of virus-growth studies in primary and passage lines of fibroblastic cells are shown in Fig. 2. These cells were relatively insusceptible to virus infection, as there was no increase in virus titer over that of the inoculum. No viral hemagglutinin was detectable in fluids of these cultures; hence, no EMD5/HA ratios could be determined. The rate of drop in titer of infectious virus in the cultures of fibroblastic cells was less than would be expected if this decrease was entirely due to heat inactivation, since heat-inactivation studies at 37 C of the PR8 strain of influenza virus in the growth medium resulted in a 5% decrease in titer in 45 min and a 9% decrease in 2.5 hr. On this basis, it was considered that the rate of infectious-virus synthesis in this system was so minimal that no rise in titer could be detected, but was sufficient to maintain titers to a slight degree above the rates of thermal inactivation. To determine the mechanisms involved in the apparently reduced susceptibility of the fibroblastic cells to PR8 influenza virus growth, a comparative study of some of the component parts of a virus growth cycle was made with the two cell systems. Attachment and irreversible binding of influenza virus to epithelioid and fibroblastic cells. Virusattachment studies were carried out at 4 C to reduce calculation errors resulting from thermal inactivation. Monolayer cultures of epithelioid and fibroblastic cells were washed in cold saline F, and virus was then added in.2-ml volumes to permit close contact of virus and cell. At various time intervals during attachment, fluids were removed from the cultures, mixed with sterile skim milk, frozen immediately, and stored at -6 C until virus assay. The multiplicity of input infection used in these experiments was.3 log1 EM5o per cell. This insured that saturation of virus receptors could not take place, and that, theoretically, 1% of the virus inoculum could be adsorbed. The percentage of virus attached was calculated as the difference between the input and the amount remaining unattached at a given time period, calculated as a percentage of the input. The results of these experiments showed that the attachment of PR8 influenza virus to epithblial-like and fibroblastic cells is nearly identical, with 8 to 85% of the virus being attached in 3 min, increasing to 95% by 5 hr. A determination of the amount of "irreversibly bound" virus was made, because any major difference in the two cell types to attach virus irreversibly would be an indication of reduced efficiency of virus infection of cells. In these experiments, "irreversibly bound" virus is defined as that virus which is not removable by RDE. An addition of 12 units of RDE in a volume of 1.5 ml to tubes containing between 16 and 2 X 16 chicken RBC, to which the PR8 strain of influenza virus had attached, was sufficient to remove 99% of the virus in 3 min at 37 C. The attachment procedure and the ratio of virus to epithelial-like and fibroblastic cells were the same as used previously. At various time periods after virus inoculation, fluids were removed from the cultures for determinations of viral attachment. The cultures were washed three times with cold saline F, and RDE prewarmed to 37 C was added. The cultures were incubated for 3 min at 37 C, and the fluids were again removed for virus assay. The amount of "irreversibly bound" virus was calculated as the difference between the inoculum and the sum of the unattached virus and that removed by RDE. The results showed that irreversible binding of virus was essentially the same with both cell types, and that the rate of this step was slower than attachment (6% irreversibly bound in 3 min when 8 to 85% of the virus had attached). Virus-growth curves with different multiplicities Downloaded from http://jb.asm.org/ on January 14, 219 by guest

VOL. 92, 1966 of infection. The previous virus-growth studies showed marked differences between fibroblastic and epithehoid cells when they were infected with low concentrations of virus. The possibility existed that the apparent resistance of the fibroblastic cells might have been due to a low level of virus production. Infection of a higher proportion of the cells initially might result in detectable increases in virus titer. The virus inoculum was added in cold saline F; attachment was allowed to take place for 9 mip at 4 C. The concentration of virus used was about 1 EU)5 per cell. At the end of the attachment period, the virus-containing fluids were removed for subsequent assay, and the cultures were washed three times in saline. An addition of 12 units of RDE at 37 C was then made, and the cultures were incubated for 3 mi at that temperature. These fluids were then removed for virus assay, the cultures were washed as before, and growth medium (FJCS) was added to the cultures. At intervals during the subsequent incubation period, tissue culture fluids and cells were removed for virus assay. The cells were removed by treatment with trypsin, frozen and thawed, and broken up by shaking with glass beads in the cold. These procedures provided the following data for cultures of both cell types: amount of inoculum attached, amount of virus irreversibly bound, the amount of virus going into the eclipse phase, and the yield of virus in fluids and cells. *-E E D 5/ml SUPERNATE - E D 5/ml CELLS 8 _ W---M HA UNITS/ml SUPERNATE _ 4-8o )--- HlA UNITS/ml CELLS HEMADSORPTION] 7,d;135-7 O 'WI CYTOTROPISM IN ANIMAL VIRUSES t \/CDW 6 -x - 3 SOLL -25 ' 5Z i / I 2-4. <~~~~~~~ O"" ~ ~ S -j 3-/ -- FlG. 3. Growth kinetics of influenza virus in epithelial-like cells. *- * E D 5/mi SUPERNATE - E D 5/mi CELLS /-9 o *----. H A UNITS/mI SUPERNATE / o----o H A UNITS/mi CELLS / A-A HEMADSORPTION / 4 8 7p 1 l 35-7 6-3 6( 5- -G( 25 Cn 4-.1 24( io!occ 34 E 'i C/) C. ~ ~ ~ ~ 8 1 E 975' a3. P 'O cr CfD z, hi - FIr. 4. Growth kinetics of influenza virus in fibroblastic cells. Figures 3 and 4 show the results of these experiments. Each point on the curves represents the mean of four samples, and each sample is a pool of material from two cultures. Also included in these figures are the results of the hemadsorption experiments described earlier, which were carried out to provide additional information regarding the capacity of fibroblastic cells to synthesize viral hemagglutinin. In the experiments with epithelioid cells (Fig. 3), the eclipse period lasted for a period of about 4 hr (2.5 hr at 37 C), and during this time only.13% of the cell-associated virus could be detected. The increase in infectious-virus titer after the eclipse period was expected from previous studies, and it was of interest to find that the titers of virus in the cells were higher than in the fluids at given time periods. the higher titers of hemagglutinin in the cells than in the fluids suggested the production of noninfectious hemagglutinin which tended to remain cell-associated. This was substantiated to some extent by finding that, at maximal titers, the loglo EID5o/HA ratios were nearly 1 log unit lower in the cells than in the fluids. The hemadsorption studies showed a rapid rise in the percentage of cells demonstrating this phenomenon after the 6th hr. The accuracy of this determination was less with the epithelial than with the fibroblastic cells after the 5% point had been reached, because the epithelioid cell boundaries were less distinct than the fibroblastic. Figure 4 -JI Downloaded from http://jb.asm.org/ on January 14, 219 by guest

976 STEWART AND FRICKEY J. BAC reriol. IL aw _j FIBROBLASTIC CULTURES: 8 *--@ E D 5/ml SUPERNATE -o E I D 5/ml CELLS _----e HA/ml SUPERNATE 7 ----o HA/ml CELLS EPITHELIAL CULTURES: *-C E D 5/ml SUPERNATE 6-O O E D 5/ml CELLS U----U HA/mi SUPERNATE O---O-C HA/ ml CELLS 5 Cn L( cl In) I~~~ virus were obtained in the cultures of epithelioid, but not in the fibroblastic, cells. The failure to 4 detect viral hemagglutinin in either cells or fluids was expected because of the low titer of virus 35 input, since a titer of about 16 EU)5o would be required to detect 1 hemagglutinin unit. These results, although compatible with the idea of - ~ epithelial-like cell contamination of fibroblastic cell cultures, still do not resolve the problem. 25 - I.- cn 2 t z -Iso -- n^ U W lwe 5 v 2 4 6 8 1 12 FIG. 5. Growth of influenza virus in epithelial-like and fibroblastic cells infected with a low-titer virus inoculum. shows the results of the same experimental procedures carried out with cultures of fibroblastic cells. The amount of virus present in the cells during the eclipse period was comparable with that obtained in epithelial-like cells, in that.19% of the cell-associated virus was present in the fibroblastic cells during the eclipse period. No significant rise in virus titer (infectious or hemagglutinin) was found in the tissue culture fluids after the eclipse period. A significant rise in these activities was found in the cells. However, the log1 EID5o/HA ratios were nearly 1 log1 lower than in epithelial cells, suggesting a greater proportion of noninfectious hemagglutinin synthesized. These results, although suggestive that fibroblastic cells can support limited replication of infectious influenza virus, are not conclusive, since a 1% level of contamination with epithelial cells would provide for the same titers of infectious virus. This level of contamination would be difficult to detect microscopically. An attempt was made to resolve this question by repeating the preceding experiments with low multiplicities of infection (.3 EID5 per cell) on the basis that the statistical probability of contaminating epithelioid cells becoming infected would be negligible. The results of these experiments are shown in Fig. 5, where it can be seen that the significant rises in titer of infectious < I DIscussIoN The results of this study clearly indicate a major difference in the efficiency of fibroblastic and epithelial-like cells derived from chick embryo lung to synthesize influenza virus strain PR8. This substantiates earlier studies (2, 16, 17, 18, 19), which also indicated the resistance of chick embryo fibroblasts to influenza virus infection. The finding, obtained with different passage levels of both cell types, that neither cell type altered appreciably in its virus-synthesizing capacity through at least six tissue culture passages offers an explanation for the findings of Stulberg and Schapira (18). These workers found a decreasing capacity of chick embryo cells to support influenza virus growth with an increase in the number of tissue culture passages. It is suggested that in their investigation the susceptible epithelioid cells were lost on passage, leaving the resistant fibroblastic cells. It was a constant finding in the present study that, unless fibroblastic cells were eliminated from epithelial-like cell cultures, the fibroblastic cells soon became the predominating population. The question of whether the use of steroid in "eliminating" the fibroblastic cells (5) from cultures of epithelioid cells had any influence on viral susceptibility was answered by a series of experiments in which prednisolone was added to cultures of both cell types prior to and during virus infection without finding any change in the results already presented. The same patterns of susceptibility and resistance have also been found when steroid was not used to aid in the selection of the epithelioid cells (16). The studies of the various aspects of influenza virus growth in the two cell types used showed no significant differences in attachment, irreversible binding, or eclipse of virus. This finding is similar to that reported by White (2), who studied growth of influenza virus in chlorioallantoic membranes of embryonated hens' eggs, which decreased in susceptibility to infection as they increased in age. Although fibroblastic cells from chick embryo lung are deficient in their capacity to synthesize infectious influenza virus, they are not incapable of synthesizing viral components. The use of the Downloaded from http://jb.asm.org/ on January 14, 219 by guest

VOL. 92, 1966 CYTOTROPISM IN ANIMAL VIRUSES 977 hemadsorption technique showed that both epithelial-like and fibroblastic cells were comparable in the rate of production of a cell-associated hemagglutinin. However, the hemagglutinin produced in the fibroblastic cell was either qualitatively or quantitatively different, or both, from that produced in epithelial-like cells, because the hemagglutinin titer obtained in fluids from mechanically disrupted cells was markedly lower in the fibroblastic cells. Furthermore, it can be assumed that the major portion of the hemagglutinin produced in fibroblastic cells is noninfectious, whereas the epithelial-like cells are characterized by production of infectious virus. It is suggested that a portion of the noninfectious hemagglutinin produced in the fibroblastic cells remains associated with cell fragments after mechanical disruption, thereby providing an explanation for the differences in hemagglutinin titer between the extracts from the two cell types. In many of the features presented in this study, the fibroblastic cells obtained from embryo lungs appear similar in their support of an abortive virus growth cycle to HeLa and L cells after infection with myxoviruses (1, 4, 8, 9, 15, 21). ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant A1-3448-3 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. BADER, J. P., AND H. R. MORGAN. 1961. A comparison of cytopathology caused by myxoviruses. I. The relation of the infectious process to cytopathology. J. Immunol. 87:8-89. 2. COLVILLE, J. M., J. M. DUNBAR, AND H. R. MOR- GAN. 1955. Temperature and host cell factors in the growth of influenza virus (PR8 strain) in avian tissue in vitro. J. Immunol. 76:264-269. 3. ERICHSEN, S., J. ENG, AND H. R. MORGAN. 1961. Comparative studies in Rous sarcoma with virus, tumor cells and chick embryo cells transformed in vitro by virus. I. Production of mucopolysaccharides. J. Exptl. Med. 114:435-44. 4. FRANKLIN, R. M., AND P. M. BREITENFELD. 1959. The abortive infection of Earle's L-cells by Fowl Plague virus. Virology 8:293-37. 5. FRANKLIN, A. E., AND J. C. SINCLAIR. 1959. Tissue culture studies with infectious hepatitis specimens. Can. J. Microbiol. 5:567-571. 6. GROSSFELD, H. 1961. Production of hyaluronic acid by fibroblasts growing from explants of Walker tumor 256: production of hyaluronidase by the tumor cells. J. Natl. Cancer Inst. 27:543-558. 7. HANKS, J. H. 1948. The longevity of chick tissue cultures without renewal of medium. J. Cellular Comp. Physiol. 31:235-26. 8. HENLE, G., A. GERARDI, AND W. HENLE. 1955. 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RINEHART, J. F., AND S. K. ABUL-HAJ. 1951. An improved method for histologic demonstration of acid mucopolysaccharides in tissue. Arch. Pathol. 52:189-194. 14. SHELOKOV, A., J. E. VOGEL, AND L. CHI. 1958. Hemadsorption (adsorption-hemagglutination) test for viral agents in tissue culture with special reference to influenza viruses. Proc. Soc. Exptl. Biol. Med. 97:82-89. 15. SMITH, D. H., AND H. R. MORGAN. 196. A cytopathogenic effect of influenza virus (PR-8 strain) for mouse fibroblastic (L) cells in vitro. J. Immunol. 85:18-189. 16. STEWART, R. B., AmN H. R. MORGAN. 1959. Studies on cytotropism in animal viruses. I. Growth of influenza virus in lung cells derived from hatched chicks and chick embryos. J. Immunol. 82:264-273. 17. STEWART, R. B., AND H. R. MORGAN. 196. Studies on cytotropism in animal viruses. II. A comparison of the growth of four influenza virus strains in vivo and in vitro in hatched chick and chick embryo lung tissue. J. Immunol. 85:465-468. 18. STULBERG, C. S., AND R. SCHAPIRA. 1953. Virus growth in tissue culture fibroblasts. I. Influenza A and herpes simplex viruses. J. Immunol. 7:51-59. 19. TYRRELL, D. A. J. 1954. New tissue culture systems for influenza, Newcastle disease and vaccinia viruses. J. Immunol. 74:293-35. 2. WHErE, D.. 1959. The mechanism of natural resistance of the allantois to influenza infection. Virology 9:68-69. 21. WILCox, W. 1959. Quantitative aspects of an in vitro virus-induced toxic reaction. I. General aspects of the reaction of Newcastle disease virus with L cells. Virology 9:3-44. Downloaded from http://jb.asm.org/ on January 14, 219 by guest