Enhancement by calcium of the detection of cytomegalovirus in cells treated with dexamethasone and dimethyl sulfoxide

P G West, W W Baker
1990 Journal of Clinical Microbiology  
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
more » ... ed, 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. 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. * Corresponding author. 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. 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. MRC-5 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 1708 on May 4, 2020 by guest
doi:10.1128/jcm.28.8.1708-1710.1990 fatcat:id2phhy4g5cw3dojtmvtjmqmb4