PLAQUE ASSAY PROCEDURE FOR COLORADO TICK FEVER VIRUS

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1 JOURNAL OF BATERIOLOGY Vol. 88, No. 1, p July, 1964 opyright a 1964 American Society for Microbiology Printed in U.S.A. PLAQUE ASSAY PROEDURE FOR OLORADO TIK FEVER VIRUS E. FRANK DEIG AND H. M. S. WATKINS Naval Biological Laboratory, School of Public Health, University of alifornia, Berkeley, alifornia Received for publication 14 February 1964 ABSTRAT DEIG, E. FRANK (University of alifornia, Berkeley), AND H. M. S. WATKINS. Plaque assay procedure for olorado tick fever virus. J. Bacteriol. 88: A reproducible plaque assay procedure is described for the quantitation of olorado tick fever virus in a cell line established from embryonic hamster tissue. Under the best conditions, plaques approximately 4 mm in diameter were formed after incubation at 37 of 4 to 6 days. Several environmental variables in the procedure were studied. Efficiency was increased markedly by combining the virus during adsorption with serum proteins, and by carrying out this step at 25 rather than at 37. The overlay medium used contained metabolites which promoted cell viability for periods greater than 1 week, and allowed plaques to develop. Plaque formation was relatively insensitive to a variation in ph between 7.1 and 8.1 (with optimal concentrations of bicarbonate). However, plaque development was inhibited with medium containing greater than.22% bicarbonate (at optimal ph), or when the initial ph was less than 7.. Investigations on olorado tick fever (TF) virus have been hampered in certain cases by the lack of an established tissue-culture plaque assay procedure for quantitation of the virus. This report presents the details of such a procedure and assesses the effect of a number of environmental variables on it. We previously described certain preliminary observations on this subject (Watkins, 1961; Deig and Watkins, 1963). MATERIALS AND METHODS The viruses used comprised seven strains (designated TF-1 through TF-7) originally isolated from human cases of TF (by the State Department of Public Health, Berkeley, alif.). These had subsequently been passaged in suckling mice and maintained as centrifuged 2% brain homogenates in normal rabbit serum. Such preparations induced cytopathogenicity in a stable cell line, termed Al, originally isolated (at hildren's ancer Research Foundation Laboratories, Boston, Mass.) from embryonic hamster tissue. ells were propagated at 37 to complete monolayers in 3-oz prescription bottles, with Eagle's minimal essential medium (MEM) plus glutamine (2 mm) and fetal calf serum (1%). Dilutions of the virus (.1 ml) were deposited directly onto the cells. Except where noted, subsequent procedures were as follows. Virus adsorption was carried out at approximately 25 for a period of 1.5 to 2 hr. The infected monolayers were then overlaid at 44 with a nutrient medium (termed HM), consisting of Earle's balanced salt solution, lactalbumin hydrolysate (.5%), yeast extract (.1 %), and bovine serum albumin (.1 %). This medium also contained fetal calf serum (2 %) and 1 % washed agar (Dulbecco and Vogt, 1954). After incubation of the monolayers at 37 for periods of 4 to 6 days, the plaques which had been formed were resolved by adding 2 ml of 1:6, (w/v) neutral red to each bottle followed by an additional incubation period of at least 4 hr prior to counting. RESULTS onditions for efficient adsorption of the virus to cells. A group of monolayers was washed twice with one of the following buffered salt solutions at ph 7.4 to 7.8: Earle's balanced salt solution plus NaHO3 (BSS), hypertonic BSS, 3 x (BSSH), or Dulbecco's buffer plus NaHO3 (DB). A second group of monolayers from the same source was similarly treated with BSS plus.5% lactalbumin hydrolysate plus.1% yeast extract (LEY) and 1% fetal calf serum, LEY plus.1% bovine serum albumin (HM), or Eagle's MEM plus 2% fetal calf serum and 2 mm glutamine. Dilutions of TF-5 virus from a single pool were made with each of these solu- 42 Downloaded from on September 5, 218 by guest

2 VOL. 88, 1964 ASSAY FOR OLORADO TIK FEVER VIRUS 43 tions and were used immediately to infect the corresponding washed monolayers. Additional variables tested with each salt solution were: adsorption temperature (4, 25, 37 ), day postinfection (4, 5) at which plaques were counted, and overlay position during incubation (i.e., normal or inverted). Typical results are presented in Table 1. TF-5 virus produced fewer and smaller plaques when corresponding dilutions of the virus were adsorbed from those buffer solutions lacking protein. Adsorption efficiency was maximal at 25, and larger plaques were formed in monolayers which had not been inverted during the incubation period. A confirmatory experiment showed also that the incorporation of fetal calf serum into the overlay medium, although serving to promote some increase in plaque size, did not affect the counts obtained from virus adsorbed under the different conditions. Serum as a plaque-promoting factor in adsorption and overlay media lacking protein. ell monolayers, washed free from residual protein with DB, were infected with TF-5 virus diluted in one of the following buffer solutions (at ph 7.4 to 7.8) supplemented with NaHO3 and 2 or 1% fetal calf serum: BSS, BSSH, DB, and Hanks' balanced salt solution (Hanks). In addition, TABLE 1. MEM, HM, and LEY media, containing 2 or 1% fetal calf serum, were also used for diluting the virus. Virus was adsorbed at 25 for approximately 2 hr. The bottles from each treatment were then placed into two groups: one group was overlaid with 1 % agar contained in the respective adsorbing media; the other, with 1 % agar in HM medium. Plaque counts were made after a 6-day incubation period. Data from this experiment (Table 2) indicated that, with HM overlay, the efficiency of plaque formation was not affected by the adsorption medium employed. Therefore, fetal calf serum was able to overcome the drop in efficiency noted previously when virus adsorption was carried out in the protein-free buffer solutions. However, no plaques were formed when these adsorption media, containing 1 % agar, were also employed as overlay. Satisfactory plaques were formed when LEY plus serum was used; however, their development was slower than with HM, necessitating a longer incubation period to avoid uiderestimating plaque numbers. Normal cell morphology was maintained in the presence of all overlay solutions, except BSSH, whose hypertonic state brought about cellular degeneration within 24 hr. Effect of ph on plaque formation. ell mono- Plaque counts obtained from the same preparation of TF virus diluted in different media and adsorbed at temperatures of 4, 25, and 37 Avg plaque count after overlay period of Adsorption medium Adsorption temp () 4 days and virus dilution of 5 days and virus dilution of LEY + 1% FS* 4 ca ca ca HM 4 ca ca ca BSS 4, 25, 37 BSSH 4, 25, 37 DB 4 ca. 5 ca ca Downloaded from on September 5, 218 by guest MEM + 2% FS 4, 25, 37 * Fetal calf serum.

3 44 DEIG AND WATKINS J. BATERIOL. TABLE 2. Plaque counts obtained from the same preparation of TF virus with different adsorption and overlay media supplemented with fetal calf serum Adsorption medium Overlay medium Avg plaque count* with virus dilution of (+2 or 1% fcs) (+2 or 1% fcs) BSS BSS BSSH BSSH DB DB LEY LEY 23 TNT TNT E HM HM 9 84 E MEM MEM Hanks Hanks BSS HM 24 ca. 2 E BSSH HM TNT E DB HM 18 ca. 1 TNT E LEY HM TNT E HM HM 15 ca. 1 TNT E MEM HM 11 ca. 1 TNT E Hanks HM 21 ca. 15 E * Plaque counts: = cells degenerated; = plaques pinpoint in size; TNT = plaques too numerous to count; E = plaques essentially confluent; = plaques fully confluent. TABLE 3. Influence of the initial ph of overlay medium on plaque counts obtained from the same preparation of TF virus Avg plaque count with overlay Virus dilution Overlay period medium at initial ph of days ca layers were washed and then infected with TF-5 virus diluted in HM. After adsorption, these were overlaid with 1% agar in an HM plus 2% fetal calf serum medium containing a fixed bicarbonate concentration (.12 %). The final ph of this medium was adjusted with 1 N NaOH or Hl to 7., 7.3, 7.6, 8.1, or 8.4. Plaque counts were made on days 4 and 6 postinfection. The initial ph of the overlay medium could fluctuate over a fairly wide range without affecting the numbers of plaque formed (Table 3). However, plaque development was observed to occur most rapidly with HM at an initial ph of 8.1. A second experiment, which utilized LEY rather than HM as an overlay medium, gave essentially similar results. However, the rate of development of plaques with this medium was depressed at the extremes of ph to such an extent that only minute forms could be seen in monolayers after 6 days of incubation. Effect of bicarbonate concentration on plaque formation. Monolayers were washed and then infected with TF-5 virus diluted in HM containing 16 ml per liter of 7.5% (w/v) NaHO3 in distilled water (final concentration,.12%). After adsorption, the monolayers were overlaid with 1 % agar in an HM plus 2% fetal calf serum medium containing a wide range of bicarbonate concentrations. Prior to the addition of agar, the ph of all solutions was adjusted to 8.1 with either 1 N NaOH or Hl. The amount of acid or base required for this purpose was shown not to affect adversely cell morphology or plaque formation. Plaques were counted after an incubation period of 4 days. The results (Table 4) indicated that bicarbonate in the overlay medium had little or no effect on these counts until a quantity in excess of 28 ml had been added. Beyond this point, a progressive reduction in numbers occurred. However, plaque size was found to be dependent on the amount of bicarbonate added to the overlay medium; with bicarbonate additions between and 2 ml per liter, plaques were small (2 mm); 4 to 8 ml, small to average (2 to 3 mm); and 12 to 16 ml, average (3 mm). oncentrations Downloaded from on September 5, 218 by guest

4 VOL. 88, 1964 ASSAY FOR OLORADO TIK FEVER VIRUS 45 of 2 to 28 ml progressively diminished plaque size. When this experiment was repeated with HM medium in the absence of calf serum, similar results were obtained, indicating that bicarbonate derived from the serum did not contribute significantly to the effects noted. Interaction of ph and bicarbonate concentration on plaque formation. Monolayers were washed, infected, and overlaid with 1% agar inhm plus 2% fetal calf serum medium containing various bicarbonate concentrations as described above. However, in this experiment the final ph of each overlay solution was not adjusted. Plaques were counted after 6 days of incubation. The results (Table 5) corresponded with those obtained from the previous experiment with two exceptions. At very low bicarbonate concentrations ( to 4 ml per liter), the acidity of the overlay medium inhibited plaque formation. Secondly, with concentrations greater than 28 ml per liter, the additional 2 days of incubation permitted the development of visible plaques not seen previously. This experiment was repeated with the use of overlay medium without fetal calf serum. The results were similar except that, under the conditions of suboptimal ph and bicarbonate concentration, plaque numbers were reduced even further. TABLE 4. Influence of bicarbonate in overlay medium with an adjusted ph on plaque counts obtained from the same preparation of TF virus Amt of 7.5% NaHOs Avg plaque countt with virus dilution of per liter of overlay medium* ml ca ca. 2 ca TNT TNT TNT TNT TNT TNT TNT ca ca. 8 ca. 1 E E E E E E E E TNT TNT TNT TNT E E E E * Overlay medium with an adjusted ph of 8.1 and containing 2% fetal calf serum. t TNT = plaques too numerous to count; E = plaques essentially confluent; = plaques fully confluent; = plaques pinpoint in size. Downloaded from TABLE 5. Influence of bicarbonate in overlay medium with a nonadjusted ph on plaque counts obtained from the same preparation of TF virus NaHOm per Initial ph of Avg plaque count with virus dilution of liter of overlay overlay medium* ml ca. 2 TNT E ca. 2 E E ca. 3 E ca. 3 E ca. 3 E ca. 3 TNT E ca ca. 1 on September 5, 218 by guest * Overlay medium with a nonadjusted ph and containing 2% fetal calf serum. t TNT = plaques too numerous to count; E = plaques essentially confluent; = confluent; = plaques pinpoint in size. plaques fully

5 46 DEIG AND WATKINS J. BATERIOL. DISUSSION This work has demonstrated that TF virus can be detected and quantitated by plaque formation in monolayers of tissue cultured Al cells. To achieve accuracy in quantitation, it is necessary to select conditions which preserve the infectivity of extracellular virus. This can be done in part by carrying out its adsorption to susceptible cells at temperatures below 37 and in media containing serum proteins. Similar findings were made by Lockhart and Groman (1958), Ushijima (1961), and others working with different virus-host cell systems. In addition, the long incubation period required for the development of visible plaques makes it necessary to apply overlay medium at a rather alkaline ph (i.e., 8.1) to prevent acidic conditions which inhibit plaque formation from being established during this time. No plaque formation occurs when an initial ph of 7., or lower, is used. A similar finding was noted by Gifford, Robertson, and Syverton (1956) with poliovirus-infected cell monolayers. Further work is needed to establish the effect of ph on the stability of TF virus and on its rate of adsorption to, and release from, susceptible cells. With certain viruses, these processes have been shown by Mclain and Hackett (1959), Taylor et al. (194), and others, to be affected markedly by ph. Plaque development is also affected by the bicarbonate concentration of the overlay medium, even under conditions of fixed initial ph. oncentrations greater than.22% bring about a progressive inhibition in numbers as well as size. The need for bicarbonate in the replication of animal viruses has been shown by Hsiung and Melnick (1958), hang (1959), and Zwartouw, Taylor-Robinson, and Westwood (196). The last group demonstrated, however, that excessive concentrations of this substance could actually inhibit virus synthesis and, in addition, bring about a significant loss of protein and nucleic acid from the infected cells (Westwood et al., 196). Inhibition of Western equine encephalomyelitis virus synthesis was shown by Lockhart and Groman (1958) to occur at bicarbonate concentrations of.22% or greater-a finding similar to our own with TF virus. A number of these experiments were repeated with the other strains of TF virus maintained in this laboratory, and confirmatory data were obtained. This suggests that, of the aspects studied, significant strain differences do not exist in the capacity for plaque formation of TF virus. AKNOWLEDGMENTS This work was sponsored by the Office of Naval Research under the terms of a contract between the Office of Naval Research and the Regents of the University of alifornia. Reproduction in whole or in part is permitted for any purposes of the United States Government. The technical assistance of Violet Nesbitt and Ronald Atwood is gratefully acknowledged. LITERATURE ITED HANG, R. S Participation of bicarbonate on RNA and protein synthesis as indicated by virus propagation in human cells. J. Exptl. Med. 19: DEIG, E. F., AND H. M. S. WATKINS Plaque assay procedure for olorado tick fever virus. Bacteriol. Proc., p DULBEO, R., AND M. VOGT Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99: GIFFORD, G. E., H. E. ROBERTSON, AND J. T. SYVERTON Propagation in vitro of polioviruses; effect of ph on virus yield and cell metabolism. Proc. Soc. Exptl. Biol. Med. 93: HSIUNG, G. D., AND J. L. MELNIK Effect of sodium bicarbonate on plaque formation of virulent and attenuated polioviruses. J. Immunol. 8: LoKHART, R. Z., AND N. B. GROMAN Some factors influencing the interaction of western equine encephalomyelitis and selected host cells. J. Infect. Diseases 13: MLAIN, M. E., AND A. J. HAKETT Biological characteristics of two plaque variants of vesicular exanthema of swine virus, type E54. Virology 9: TAYLOR, A. R., D. G. SHARP, D. BEARD, AND J. W. BEARD Influence on ph on the molecular stability of the equine encephalomyelitis virus protein (Eastern strain). J. Infect. Diseases 67: USHIJIMA, R. N Plaque mutants of western equine encephalitis virus, p. 62. Ph.D. Thesis, University of Utah, Salt Lake ity. WATKINS, H. M. S Studies on viral aerosols: survival and infectivity of aerosolized olo- Downloaded from on September 5, 218 by guest

6 VOL. 88, 1964 ASSAY FOR OLORADO TIK FEVER VIRUS 47 rado tick fever and vesicular stomatitis viruses. Tech. Progr. Rept. 31, Nav. Biol. Lab., Univ. alifornia, p WESTWOOD, J.. N., G. ALEYARD, D. TAYLOR- ROBINSON, AND H. T. ZWARTOUW The production of high titer poliovirus in concentrated suspensions of tissue cultured cells. Brit. J. Exptl. Pathol. 41: ZWARTOUW, H. T., D. TAYLOR-ROBINSON, AND J.. N. WESTWOOD Growth of hightiter poliovirus in nutrient-free saline suspensions of ERK cells. Virology 1: Downloaded from on September 5, 218 by guest