Vol. 28, No. 8
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1990, p. 1708-1710
0095-1137/90/081708-03$02.00/0 Copyright C 1990, American Society for Microbiology
Enhancement by Calcium of the Detection of Cytomegalovirus in Cells Treated with Dexamethasone and Dimethyl Sulfoxide PATRICIA G. WESTl.2* AND WILBER W. BAKER2 SmithKline Beecham Clinical Laboratories, Norristown, Pennsylvania 19403,1 and Villanova University,
Villanova, Pennsylvania 190852 Received 27 February 1990/Accepted 14 May 1990
MRC-5 cultures treated with dexamethasone, dimethyl sulfoxide, and calcium were compared with untreated cultures and cultures treated with dexamethasone and dimethyl sulfoxide for the detection of human cytomegalovirus by the shell vial method. The addition of calcium resulted in significantly increased growth of human cytomegalovirus AD169. Protein studies and ceil counts showed renewed cell proliferation in the treated cultures. When low-titer clinical specimens were retested, 27.5%O of all positives revealed were found only on the vials treated with calcium. In a group of freshly tested specimens, the calcium-treated group accounted for 20% of the positives.
Finally, we tested a number of clinical specimens with both untreated cells and cells treated with thé various additives to determine any effect on the rate of detection of positives and on the speed and facility of reading these specimens.
One of the earliest interactions between human cytomegalovirus (HCMV) and its host cell is marked by the formation of a specific antigen product of the immediate early viral genes which is found exclusively in the host cell nucleus. Detection of this antigen has been made possible by the development of specific monoclonal antibodies, and in combination with the shell vial culture, it is very useful in determining the presence of HCMV in clinical specimens from patients suspected of having this infection. However, as with all HCMV culture methods, the sensitivity of this technique is dependent upon the metabolic state of the host cells as well as the amount of viable virus present in the specimen. The fact that actively growing cells are most effective for the detection of HCMV is now recognized (1-3); however, most virology reference laboratories are constrained to use commercial cultures that have reached confluency several days before use and are no longer actively metabolizing. At this stage, the cultures have been shown to be well past the stage allowing optimum viral growth (2). We have previously demonstrated that the addition of dexamethasone (DEX) and dimethyl sulfoxide (DMSO) to the culture medium used with MRC-5 host cells has enhanced the detection of HCMV in these cells (4). In this study, we investigate the effect on viral growth of the addition of calcium, a growth factor which is known to stimulate the proliferation of cells in quiescent cultures (D. P. Chopra, J. K. Sullivan, and S. Reece-Kooger, In Vitro 24:222A, 1988; V. J. Cristolfalo, T. Sorger, G. Gerhard, and P. D. Phillips, In Vitro 24:141A, 1988). We tested calcium both alone and in combination with DEX and DMSO and determined the effect on cellular growth by measuring increases in cellular protein and in the number of cells present in each vial. Increases in these parameters in vials treated with the agents would indicate that the renewed cell growth is a result of the treatments. A correlation between increased cell growth and increased viral detection would serve to reinforce previous investigations linking actively growing cells with enhanced viral production (1-3). We report, in addition, the effect of calcium on actual virion production and on the detection of early viral proteins through immunostaining, the method used to detect virus in clinical specimens. *
MATERIALS AND METHODS Stock virus, cell cultures, and reagents. Stock cultures of HCMV (strain AD169) were prepared by growing the virus in MRC-5 cultures, disrupting the cells by vortexing with glass beads, centrifuging to precipitate cell debris, and diluting the supernatant to the required concentration. MRC5 cultures grown on cover slips in shell vials were obtained from Whittaker Bioproducts, Walkersville, Md. Eagle minimum essential medium containing 2% fetal bovine serum, 1% glutamine, 50 mg of gentamicin per liter, 50 mg of vancomycin per liter, and 2.5 mg of amphotericin B (Fungizone) per liter was used throughout. Medium, serum, and gentamicin were obtained from Whittaker. Vancomycin was obtained from Sigma Chemical Co., St. Louis, Mo., and glutamine and amphotericin B were obtained from flow Laboratories, McLean, Va. DMSO, DEX, and calcium chloride were obtained from Sigma. Mouse anti-immediate early CMV antibodies were obtained from Dupont-Biotech Research Laboratories, Rockville, Md. Goat anti-mouse immunoglobulin G fluorescein conjugate was obtained from Tago, Inc., Burlingame, -Calif. DEX was initially dissolved in ethanol and then diluted in medium to 10-' M. DMSO was used at a 1% concentration in medium. Calcium chloride was initially dissolved in water and then diluted in medium to a concentration of 2 mM. Test methods. The shell vial test and methods for the determination of virus susceptibility and growth in treated and untreated cells were as previously described (4). The various treatments used fall into the following categories: (i) medium only (control group); (ii) medium plus 2 mM calcium (calcium group); (iii) medium plus i0-5 M DEX and 1% DMSO (DEX-DMSO group); (iv) medium plus DEX, DMSO, and 2 mM calcium (DEX-DMSO-Ca group). Before inoculation with virus, the first two groups described above contained the medium in which the vials were shipped, while the second two groups were pretreated for 24 h with medium containing 10-5 M DEX. The four treatment groups were
Corresponding author. 1708
CALCIUM-MEDIATED ENHANCEMENT OF CMV
VOL. 28, 1990 -6.3
0-6.5-
Q
10-5.5 o
Q0
200 180'
O
c
10-6.0.
1709
0 Co
160'a
m
140'
°> 120' E 100'
-5.0
10-50. -4.5
ER
104.5 10
S
g
E z
10-4.0
10-3.5
used both in the centrifugation step and during the subsequent 20-h incubation.
Determination of virus production. To determine the effect of the various cell treatments 'on total virion production, confluent MRC-5 vials were divided into the four treatment groups (five vials each) and treated as described above. The four groups were then inoculated with HCMV AD169 at a multiplicity of infection of 10, centrifuged for 1 h, incubated for 5 days in the presence of the various treatment media, and harvested. The viral suspension from each treatment group was titrated from 1'0 through 10-8, 4nd each dilution was tested on six untreated MRC-5 vials. A 50% endpoint or 50% tissue culture infective dose for each treatment group was calculated by the Reed-Muench method. Virus susceptibility study. In order to ascertain the effect of the cell'treatments on the detection of immediate-early viral proteins, an HCMV AD169 multiplicity of infection of 0.01, which approximates the concentration of virus in a clinical specimen, was tested on cells- in the four treatment groups. Infectious centers were counted and averaged for each
20 '
16 Calcum
Dex-DMSO
DEX-OMSOCalci m
Cell
Treatments
FIG. 2. Typical HCMV infectious centers, as determined by fluorescein-stained nuclei, were counted for each treatment group. Four experiments, each with six cover slips per treatment group, were averaged to obtain the figures shown. HCMV AD169 at a multiplicity of infection of 0.01 was used. Statistical analysis (t test) showed significant differences between treatment with DEXDMSO-Ca and no treatment (P < 0.01), treatment with calcium alone (P < 0.01), and treatment with DEX-DMSO (P < 0.05).
A second study was done by comparing HCMV detection cells pretreated with DEX and posttreated with' DXDMSO (the combination then used in our laboratory) and that on cells pretreated with DEX and posttreated with DEX, DMSO, and 2 mM calcium. These specimens were tested immediately on receipt by the laboratory. on
RESULTS When HCMV stock virus AD169 was grown in untreated cells and in cells that were treated with the four additive groups, almost 100-fold more virus was produced in the
'a o c
0
.2
group.
Total protein determination. Protein was determined by washing the cells with Hanks buffered saline solution, solubilizing them with 0.5 ml of 0.1 M NaOH, adding 2 ml of Coomassie blue solution (Pierce Chemical Co., Rockford, Ill.), and reading the A630 after 15 min. An albumin standard (Pierce) was also diluted in 0.1 M NaOH, and protein concentrations were determined from a nonlinear standard curve (Gilford Response). Values for the six vials of each group we're averaged. Cell number determination. Cell counts were made with a hemacytometer on three vials per treatment group, and the three counts were averaged for the recorded value. Clinical specimen study. Clinical specimens obtained from a variety of sources were submitted to our facility for the isolation of HCMV. Previously tested HCMV-positive specimens were stored at 4°C for 1 to 3 weeks (there may have been loss of infectivity during this time) and then tested on cèlls from three of the treatment groups. The methods were as outlined previously (4), with the exception that treatment medium was used in the centrifugation step.
60' 40' None
Calcim
Coil Treatments FIG. 1. Two separate viral titrations were run with six cover slips per dilution, and 50% tissue culture infective doses (TCID50s) were determined for each treatment group by the Reed-Muench method. Results were averaged, and the log values of the TCID50s were graphed in the above chart. An analysis of variance specific post hoc test showed that the treatment with DEX-DMSO-Ca differed from all other treatments (P < 0.05) (5).
80'
c
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1990, p. 1708-1710
0095-1137/90/081708-03$02.00/0 Copyright C 1990, American Society for Microbiology
Enhancement by Calcium of the Detection of Cytomegalovirus in Cells Treated with Dexamethasone and Dimethyl Sulfoxide PATRICIA G. WESTl.2* AND WILBER W. BAKER2 SmithKline Beecham Clinical Laboratories, Norristown, Pennsylvania 19403,1 and Villanova University,
Villanova, Pennsylvania 190852 Received 27 February 1990/Accepted 14 May 1990
MRC-5 cultures treated with dexamethasone, dimethyl sulfoxide, and calcium were compared with untreated cultures and cultures treated with dexamethasone and dimethyl sulfoxide for the detection of human cytomegalovirus by the shell vial method. The addition of calcium resulted in significantly increased growth of human cytomegalovirus AD169. Protein studies and ceil counts showed renewed cell proliferation in the treated cultures. When low-titer clinical specimens were retested, 27.5%O of all positives revealed were found only on the vials treated with calcium. In a group of freshly tested specimens, the calcium-treated group accounted for 20% of the positives.
Finally, we tested a number of clinical specimens with both untreated cells and cells treated with thé various additives to determine any effect on the rate of detection of positives and on the speed and facility of reading these specimens.
One of the earliest interactions between human cytomegalovirus (HCMV) and its host cell is marked by the formation of a specific antigen product of the immediate early viral genes which is found exclusively in the host cell nucleus. Detection of this antigen has been made possible by the development of specific monoclonal antibodies, and in combination with the shell vial culture, it is very useful in determining the presence of HCMV in clinical specimens from patients suspected of having this infection. However, as with all HCMV culture methods, the sensitivity of this technique is dependent upon the metabolic state of the host cells as well as the amount of viable virus present in the specimen. The fact that actively growing cells are most effective for the detection of HCMV is now recognized (1-3); however, most virology reference laboratories are constrained to use commercial cultures that have reached confluency several days before use and are no longer actively metabolizing. At this stage, the cultures have been shown to be well past the stage allowing optimum viral growth (2). We have previously demonstrated that the addition of dexamethasone (DEX) and dimethyl sulfoxide (DMSO) to the culture medium used with MRC-5 host cells has enhanced the detection of HCMV in these cells (4). In this study, we investigate the effect on viral growth of the addition of calcium, a growth factor which is known to stimulate the proliferation of cells in quiescent cultures (D. P. Chopra, J. K. Sullivan, and S. Reece-Kooger, In Vitro 24:222A, 1988; V. J. Cristolfalo, T. Sorger, G. Gerhard, and P. D. Phillips, In Vitro 24:141A, 1988). We tested calcium both alone and in combination with DEX and DMSO and determined the effect on cellular growth by measuring increases in cellular protein and in the number of cells present in each vial. Increases in these parameters in vials treated with the agents would indicate that the renewed cell growth is a result of the treatments. A correlation between increased cell growth and increased viral detection would serve to reinforce previous investigations linking actively growing cells with enhanced viral production (1-3). We report, in addition, the effect of calcium on actual virion production and on the detection of early viral proteins through immunostaining, the method used to detect virus in clinical specimens. *
MATERIALS AND METHODS Stock virus, cell cultures, and reagents. Stock cultures of HCMV (strain AD169) were prepared by growing the virus in MRC-5 cultures, disrupting the cells by vortexing with glass beads, centrifuging to precipitate cell debris, and diluting the supernatant to the required concentration. MRC5 cultures grown on cover slips in shell vials were obtained from Whittaker Bioproducts, Walkersville, Md. Eagle minimum essential medium containing 2% fetal bovine serum, 1% glutamine, 50 mg of gentamicin per liter, 50 mg of vancomycin per liter, and 2.5 mg of amphotericin B (Fungizone) per liter was used throughout. Medium, serum, and gentamicin were obtained from Whittaker. Vancomycin was obtained from Sigma Chemical Co., St. Louis, Mo., and glutamine and amphotericin B were obtained from flow Laboratories, McLean, Va. DMSO, DEX, and calcium chloride were obtained from Sigma. Mouse anti-immediate early CMV antibodies were obtained from Dupont-Biotech Research Laboratories, Rockville, Md. Goat anti-mouse immunoglobulin G fluorescein conjugate was obtained from Tago, Inc., Burlingame, -Calif. DEX was initially dissolved in ethanol and then diluted in medium to 10-' M. DMSO was used at a 1% concentration in medium. Calcium chloride was initially dissolved in water and then diluted in medium to a concentration of 2 mM. Test methods. The shell vial test and methods for the determination of virus susceptibility and growth in treated and untreated cells were as previously described (4). The various treatments used fall into the following categories: (i) medium only (control group); (ii) medium plus 2 mM calcium (calcium group); (iii) medium plus i0-5 M DEX and 1% DMSO (DEX-DMSO group); (iv) medium plus DEX, DMSO, and 2 mM calcium (DEX-DMSO-Ca group). Before inoculation with virus, the first two groups described above contained the medium in which the vials were shipped, while the second two groups were pretreated for 24 h with medium containing 10-5 M DEX. The four treatment groups were
Corresponding author. 1708
CALCIUM-MEDIATED ENHANCEMENT OF CMV
VOL. 28, 1990 -6.3
0-6.5-
Q
10-5.5 o
Q0
200 180'
O
c
10-6.0.
1709
0 Co
160'a
m
140'
°> 120' E 100'
-5.0
10-50. -4.5
ER
104.5 10
S
g
E z
10-4.0
10-3.5
used both in the centrifugation step and during the subsequent 20-h incubation.
Determination of virus production. To determine the effect of the various cell treatments 'on total virion production, confluent MRC-5 vials were divided into the four treatment groups (five vials each) and treated as described above. The four groups were then inoculated with HCMV AD169 at a multiplicity of infection of 10, centrifuged for 1 h, incubated for 5 days in the presence of the various treatment media, and harvested. The viral suspension from each treatment group was titrated from 1'0 through 10-8, 4nd each dilution was tested on six untreated MRC-5 vials. A 50% endpoint or 50% tissue culture infective dose for each treatment group was calculated by the Reed-Muench method. Virus susceptibility study. In order to ascertain the effect of the cell'treatments on the detection of immediate-early viral proteins, an HCMV AD169 multiplicity of infection of 0.01, which approximates the concentration of virus in a clinical specimen, was tested on cells- in the four treatment groups. Infectious centers were counted and averaged for each
20 '
16 Calcum
Dex-DMSO
DEX-OMSOCalci m
Cell
Treatments
FIG. 2. Typical HCMV infectious centers, as determined by fluorescein-stained nuclei, were counted for each treatment group. Four experiments, each with six cover slips per treatment group, were averaged to obtain the figures shown. HCMV AD169 at a multiplicity of infection of 0.01 was used. Statistical analysis (t test) showed significant differences between treatment with DEXDMSO-Ca and no treatment (P < 0.01), treatment with calcium alone (P < 0.01), and treatment with DEX-DMSO (P < 0.05).
A second study was done by comparing HCMV detection cells pretreated with DEX and posttreated with' DXDMSO (the combination then used in our laboratory) and that on cells pretreated with DEX and posttreated with DEX, DMSO, and 2 mM calcium. These specimens were tested immediately on receipt by the laboratory. on
RESULTS When HCMV stock virus AD169 was grown in untreated cells and in cells that were treated with the four additive groups, almost 100-fold more virus was produced in the
'a o c
0
.2
group.
Total protein determination. Protein was determined by washing the cells with Hanks buffered saline solution, solubilizing them with 0.5 ml of 0.1 M NaOH, adding 2 ml of Coomassie blue solution (Pierce Chemical Co., Rockford, Ill.), and reading the A630 after 15 min. An albumin standard (Pierce) was also diluted in 0.1 M NaOH, and protein concentrations were determined from a nonlinear standard curve (Gilford Response). Values for the six vials of each group we're averaged. Cell number determination. Cell counts were made with a hemacytometer on three vials per treatment group, and the three counts were averaged for the recorded value. Clinical specimen study. Clinical specimens obtained from a variety of sources were submitted to our facility for the isolation of HCMV. Previously tested HCMV-positive specimens were stored at 4°C for 1 to 3 weeks (there may have been loss of infectivity during this time) and then tested on cèlls from three of the treatment groups. The methods were as outlined previously (4), with the exception that treatment medium was used in the centrifugation step.
60' 40' None
Calcim
Coil Treatments FIG. 1. Two separate viral titrations were run with six cover slips per dilution, and 50% tissue culture infective doses (TCID50s) were determined for each treatment group by the Reed-Muench method. Results were averaged, and the log values of the TCID50s were graphed in the above chart. An analysis of variance specific post hoc test showed that the treatment with DEX-DMSO-Ca differed from all other treatments (P < 0.05) (5).
80'
c
