An 80- to 150-ml amount of calf or simian rotavirus-containing cell culture harvests of MA-104 cells were treated with 50 jig of trypsin per ml and hydroex-.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1980, p. 1133-1135
Vol. 40, No. 6
0099-2240/80/12-1133/03$02.00/0
Rotavirus Concentration from Cell Culture Harvests: Trypsin Treatment Followed by Hydroextraction SAMI RAMIA AND SYED A. SATTAR* Department of Microbiology and Immunology, School of Medicine, University of Ottawa, Ottawa, Ontario KIN 9A9, Canada
An 80- to 150-ml amount of calf or simian rotavirus-containing cell culture harvests of MA-104 cells were treated with 50 jig of trypsin per ml and hydroextracted overnight (4°C) with polyethylene glycol 6,000. The concentrate was resuspended in 8 to 10 ml of tryptose phosphate broth and plaque assayed. Between 85 and 97% of the input virus could be recovered with a concentration of up to 15-fold.
Earlier work in our laboratory showed that polyethylene glycol hydroextraction is a useful method for the second-step concentration of entero- (9), reo- (9), and rotaviruses (11) from the water environment. Because of the relatively simple and efficient nature of this technique, we attempted to extend its use to the concentration of rotaviruses from cell culture harvests (CCH). This communication describes the findings of these experiments. Trypsin (1:250; GIBCO, Grand Island, N.Y.) and soybean trypsin inhibitor (Sigma Chemical Co., St. Louis, Mo.) were prepared separately as 0.1% stock solutions in Earle balanced salt solution. They were passed through a 0.22-,um membrane filter before storage in 1.0-ml volumes at -20°C. Polyethylene glycol 6,000 powder was purchased from J. T. Baker Chemical Co., Phillipsburg, N.J. MA-104 cells were used throughout this study. The cells were routinely cultivated as monolayers in 75-cm2 plastic tissue culture flasks (Flow Laboratories, Rockville, Md.) with Eagle minimal essential medium in Earle base (Autopow; Flow Laboratories). Each 450-ml portion of the medium was supplemented with 25 mg of gentamicin (Schering Corp., Bloomfield, N.J.), 13.5% of a 5.6% solution of sodium bicarbonate, 5.0 ml of a 200 mM solution of L-glutamine, and 50 ml of virus- and mycoplasma-tested fetal bovine serum (Microbiological Associates, Bethesda, Md.). Other details for the cultivation, maintenance, and passage of the cells have been described elsewhere (10). Simian rotavirus SA-11 (strain H96) was kindly supplied to us by H. Malherbe of the University of Texas at San Antonio. Strain C486 of the calf rotavirus was a gift from L. Babuik of the University of Saskatchewan at Saskatoon. These viruses were plaque purified in MA-104 cells in our laboratory, and the same cells were
used for the preparation of the CCH used in this study. Because fetal bovine serum has been found to be inhibitory for rotaviruses (1), it was necessary to wash the cell monolayers twice with Earle balanced salt solution before virus inoculation. After allowing the virus to adsorb for 1 h at 37°C, maintenance medium (Eagle minimal essential medium without serum and with 10,ug of trypsin per ml) was introduced in the cultures, and they were placed back at 37°C. When nearly 75% of the monolayer was affected by virus cytopathic effects, the cultures were frozen (-20°C) and thawed three times. After centrifugation at 1,000 x g for 15 min, the supernatant (CCH) was kept frozen at -80°C until ready to be used. To the CCH, trypsin was added to a final concentration of 50 ,g/ml. The trypsin-containing CCH was then left at 37°C for 1 h. This was followed by the addition of trypsin inhibitor to the CCH to a final concentration of 100 ,ug/ml and an additional incubation period of 30 min at 37°C. After removing a 5-ml amount to serve as a control, the remaining CCH was placed in a dialysis sac (2.7-cm diameter, 4.8-nm pore size) purchased from Fisher Scientific Co., Pittsburgh, Pa. The length of tubing required for a 100-ml volufie of CCH was approximately 30 cm. For hydroextraction, the sac was placed in a 250-ml glass beaker. About 200 g of polyethylene glycol 6,000 powder was required to evenly cover the sacs inside the beaker. The beaker was kept overnight at 4°C. Nearly all of the liquid from inside the sacs was hydroextracted by the next morning. The material remaining inside each sac was suspended in 8 to 10 ml of lx tryptose phosphate broth at pH 7.2 and plaque assayed (10) after passage through a 0.22-,um membrane filter. In the initial experiments, CCH was hydroextracted without any prior treatment. It was
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APPL. ENVIRON. MICROBIOL.
NOTES
found that over 99% of the input plaque-forming units (PFU) became undetectable in the concentrates. However, with prior trypsin treatment and polyethylene glycol hydroextraction, a 10to 15-fold concentration of calf rotavirus CCH could be achieved with an 85 to 94% recovery of the input PFU (Table 1). Similar experiments were subsequently performed with simian rotavirus SA-1l, and the data from these experiments are summarized in Table 2. With a 10-fold concentration, nearly 87 to 97% of the input PFU could be recovered. The only pretreatment of the sample required in this technique is its exposure to trypsin. Without such enzyme pretreatment, inhibitory activity accumulated in the hydroextracted material TABLE 1. Concentration of calf rotavirus from MA104 CCH by using trypsin treatment and hydroextractiona Expt no.
1 2 3 4 5
Initial vol of CCH (ml)
80 80 80 150 150
Vol of
Total PFU
(x109)
concentrate
in con-
in CCH
(Ml)
centrate
2.25 2.25 2.70 5.10 5.00
8 8 8 10 10
2.00 2.10 2.30 4.40 4.80
Total PFU
(x109)
% Recovery
89 93 85 86 96
9 3.12 90 3.46 108 Trypsin was added to CCH to a final concentration of 50 Itg/ml. After 1 h of incubation at 370C, trypsin inhibitor was added to the trypsin-containing CCH to 100 and an additional a final concentration of ltg/ml, incubation period of 30 min at 37°C was allowed. Then the CCH was placed in a dialysis sac and hydroextracted with polyethylene glycol 6,000 overnight at 4°C. The material remaining in the sac was resuspended in 8 to 10 ml of tryptose phosphate broth (pH 7.2) and plaque assayed in MA-104 cells after membrane filtration.
Mean a
TABLE 2. Concentration of simian rotavirus SA-1I from MA-104 CCH by using trypsin treatment and
hydroextractiona Expt no.
1 2 3 4
(ml)
Total PFU (xI0) in CCH
80 80 80 100
4.00 3.70 3.70 4.60
Initial vol of CCH
4.00 85 Mean a See Table 1, footnote a.
Total
Vol of
PU8
concen-
trate
.
% Re covery
(ml)
centrate
8 8 8 10
3.60 3.40 3.60 4.00
90 92 97 87
3.65
92
8.5
produced a pronounced loss of virus infectivity. Although the exact nature of this virus inhibitory component(s) remains to be elucidated, its susceptibility to trypsin indicates that it is proteinaceous. Resuspension of the concentrated CCH in tryptose phosphate broth was necessary to avoid virus loss by adsorption during its passage through 0.22-,um membrane filters (12). Proteins such as those in tryptose phosphate broth are also known to help in the deaggregation of virus clumps (5). A comparative study of a variety of proteinaceous solutions had shown tryptose phosphate broth to be noninhibitory to rotaviruses (11). Addition of polyethylene glycol is known to precipitate certain types of viruses from CCH, and this phenomenon has been applied to their concentration (6). Such a technique may not be applicable to those viruses which are inhibited in the presence of polyethylene glycol (8). Negatively charged membrane filters have previously been used for the concentration of enteroviruses from CCH (7, 12). Because the presence of membrane-coating components in the CCH interfered with virus adsorption to the filtration matrix, it was necessary to subject the sample to a series of time-consuming pretreatments. Furthermore, the adsorption of viruses to and their elution from the filters required the use of pH levels which are now known to be deleterious to rotaviruses (2, 3). Although the membrane filtration technique was subsequently modified to allow its use with rotaviruses (3), it still required a considerable degree of sample treatment before and after its passage through the filters. Prefiltration of the sample and adjustment of its pH to 6.0 was found to be necessary for the concentration of influenza virus-containing allantoic fluid with electropositive membrane filters (4). The concentration factor achieved by this technique was in the same range as reported here for polyethylene glycol hydroextraction. From the preliminary data presented here, polyethylene glycol hydroextraction of trypsinpretreated CCH appears to be a relatively simple and efficient means of rotavirus concentration. This should be particularly helpful since cell culture-adapted members of this group generally do not grow to high titers. Attempts are now being made to extend this technique to other viruses as well as larger volumes of CCH. This investigation was supported by grant PR 911 from the Ontario Ministry of Health. The secretarial assistance of Monique D'Amour is gratefully acknowledged.
VOL. 40,1980 LrIERATURE CITED 1. Clark, S. IL, B. B. Barnett, and R. S. Spendlove. 1979. Production of high-titer bovine rotaviuvs with trypsin. J. Clin. Microbiol. 9:413-417. 2. Estes, K., D. Y. Graham, E. ML Smith, and C. P. Gerba. 1979. Rotavirus stability and inactivation. J. Gen. ViroL 43:403-409. 3. Farrah S. R, S. M. Goyal, C. P. Gerba, RI Coaklin, C. Wallis, J. L. Melnick,, and H. L; DuPont. 1978. A simple method for concentration of enterovirues and rotaviruses from cell culture harvests using membrane filters. Intervirology 9:56-59. 4. Goyal, S. AL, H. Hanssen, and C. P. Gerba. 1980. Simple method for the concentration of influenza virus from allantoic fluid on microporous filters. Appl. Environ. Microbiol. 39:500-604. 5. Hamblet, F. E., W. L Hill Jr, and E. W. Akin. 1967. Effect of plaque assay diluent upon enumeration of poIiovirus type 1. Appl. Microbiol. 15:208. 6. Hamlin, C., and G. Lusier. 1979. Concentration of human cytomegalovirs from large volumes of tissue culture fluids. J. Gen. Virol. 42:193-197.
NOTES
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7. Henderson, M., C. Wallis, and J. I Melnick. 1976. Concentration and purification of enteroviruses by membrane chromatography. Appl. Environ. Microbiol. 32:6894693. 8. Lotekeva, V. J., M. P. Chumakov, V. . Zhevandrova, and L. A. Shekoyan. 1973. Differential sensitivity of virulent and attenuated strains of type 1 poliovirus to polyethylene glycol as a possible new genetic marker. Arch. Gesamte Virusforsch. 41:155-159. 9. Ramia, S., and S. A. Sattar. 1979. Second-step concentration of viuses in drinking and surface waters using polyethylene glycol hydroextraction. Can. J. Microbiol. 25:587-592. 10. Ramia, S., and S. A. Sattar. 1979. Simian rotavirus SA11 plaque formation in the presence of trypsin. J. Clin. Microbiol. 10:609-614. 11. Ramia, S., and S. A. Sattar. 1979. Concentration of seeded simian rotavirus SA-11 from potable waters by using talc-Celite layers and hydroextraction. Appl. Environ. Microbiol. 39:493-499. 12. Waflis, C., and J. L Melnick. 1967. Concentration of enterovinrses on membrane filters. J. Virol. 1:472-477.
