Journal of Virological Methoak, 39 (1992) 3 1 l-3 18 0
1992 Elsevier Science Publishers
311
B.V. / All rights reserved / 0166-0934/92/$05.00
VIRMET 01390
Comparison of MRC-5 and continuous cell lines for detection of cytomegalovirus in centrifugation cultures Marie-Christine Mazeron, Badia Benjelloun, Claire Bertrand, Jean-Luc Pons and Yvonne PQol Service de BactPriologie-Virologie.
H6pital Saint-Louis,
UniversitP Paris VII, Paris (France)
(Accepted 5 May 1992)
Summary
Continuous cell lines were assessed for use for rapid human cytomegalovirus (HCMV) detection procedures combining tissue culture, centrifugation, and immediate early antigen (IEA) immunostaining. Human cells (MRC-5 embryonic fibroblasts, U373MG astrocytoma cells, differentiated teratocarcinoma (Tera-2) cells), murine cells (BALB/c-3T3 and Y-l cells), BHK:!r hamster cells, and mink lung (ML) cells were first inoculated with HCMV laboratory strain. IEA synthezising cells were detected by immunoperoxidase assay using a monoclonal antibody. ML cells and differentiated Tera-2 cells exhibited more positive cells than MRC-5 cells. BHK 21, and MRC-5 cells were equivalent in sensitivity whereas U-373MG, BALB/c-3T3, and Y-l cells had only reduced IEA positive cells. When 63 urine specimens were inoculated onto MRC-5, ML and differentiated Tera-2 cells, 20 (31.7%) were positive in MRC-5 cells versus 18 (28.5%) in ML or Tera-2 cells. Moreover, greater numbers of infected cells were detected in MRC-5 cells than in these two cell lines. MRC-5 cells were superior for detection of HCMV in clinical samples by centrifugation cultures. Cytomegalovirus (human); Centrifugation culture; Cell lines, continuous; Fibroblast (human); Immediate early antigen
Correspondence to: M.C. Mazeron, Service de Bactkiologie-Virologie, Ambroise Park, 7’5475,Paris Ctdex 10, France.
HBpital LariboisDre, 2, rue
312
Introduction Human cytomegalovirus (HCMV) is highly restricted in its host range in cell cultures. Efficient replication and release of high titers of progeny virions are obtained only in human diploid tibroblasts (HF). Other human cell types tested, such as epithelial cells (Smith, 1986; Tanaka et al., 1984), endothelial cells (Smiley et al., 1988), rabdomyosarcoma cells (Tanaka et al., 1985) are moderately permissive. Human teratocarcinoma stem cells are non-permissive for HCMV but become permissive after being induced to differentiate by treatment with retinoic acid (RA) (Goncziil et al., 1984; LaFemina and Hayward, 1986). In non-permissive cells, failure in progression of the lytic cycle occurs at any of the steps between adsorption and virion production (LaFemina and Hayward, 1988). HCMV immediate early antigen (IEA) expression does occur in some non-permissive xenogeneic cell lines. HCMV isolation procedures are usually carried out using HF. Rapid methods use a combination of low-speed centrifugation of the clinical specimens onto cell monolayers and detection of IEA in infected cells (Mazeron et al., 1984; Gleaves et al., 1984). As these methods do not necessitate a complete viral replication cycle, continuous cell lines which allow expression of IEA following infection by HCMV strains could be used for diagnosis. These would offer advantages for the clinical laboratory compared to HF, which are generally useful only over a limited number of passages. Mink lung (ML) cell cultures were found to be more sensitive than HF for detection of HCMV strains (MacKenzie and McLaren, 1989). In this study, human and non-human cell cultures were compared for the degree of sensitivity to IEA expression of HCMV reference and wild strains.
Materials and Methods Cell cultures The sources of the cell lines used were as follows: MRC-5 human embryonic lung tibroblasts, passages 28 to 36, (Bio-Merieux); I-I-373MG human astrocytoma cells, (J-L Virelizier, Institut Pasteur, Paris); Tera-2 human teratocarcinoma stem cells, BALB/c-3T3 established murine cells, BHK*i(C-13) established hamster kidney cells and ML cells (G Peries, Paris, Inserm U-107); Y-l murine adrenal tumor cells (American Type Culture Collection, ATCC CCL 79). Y-l cells were cultured in Ham’s FlO medium containing 15% horse serum and 2.5% fetal bovine serum and the other cells in Eagle’s Minimal Essential Medium (MEM) with 10% fetal bovine serum. Differentiation of Tera-2 cells was achieved by subculture in the same medium containing RA (Aldrich) at a concentration of 10v5 M for 10 days. Cell monolayers grown in 24-well plates were used for HCMV isolation. The number of cells seeded in each well was adjusted to obtain subconfluent
313
monolayers within 48 to 72 h for MRC-$24
and 48 h for the other cell types.
HCMV reference strain
The AD169 strain of HCMV (ATTC VR-538) was propagated in MRC-5 cell cultures. The cells were inoculated at a multiplicity of infection of one. When cytopathic effects reached 90%, supernatants were saved, clarified by low speed centrifugation and retained as virus stocks. These stocks were dispensed in l-ml aliquots and stored at -80°C. Immediately before use, a stock aliquot was thawed and diluted serially IO-fold. Clinical specimens and processing
Routine urine specimens submitted for HCMV cultures were obtained from marrow and renal transplant recipients and patients with the acquired immunodeficiency syndrome. The specimens were collected in an equal volume of transport medium (MEM containing 10% fetal bovine serum, sorbitol and antibiotics) and kept on ice during transportation to the virology laboratory. For processing specimens, 5 ml of each urine sample was filtered through a 0.45~pm pore size filter (Sartorius). After inoculation, all specimens were stored at -80°C. Rapid detection of CMV Inoculation and incubation of specimens. Immediately before inoculation, the culture medium was decanted from the cell monolayers in the plates. 0.1 ml of diluted viral stock was added to each of four wells and 0.5 ml of processed urine samples were inoculated into each of two wells. The supernatant volume in each well was made up to 1 ml with maintenance medium. Inoculated 24-well plates were centrifuged at 2900 x g at 36°C for 45 min. Inocula were then removed and replaced by fresh medium. The cells were incubated at 37°C in 5% COZ atmosphere for 24 or 48 h. Fixation and staining procedures. Cells were fixed in a mixture of cold acetone and distilled water (9: 1, v/v) at -20°C for 10 min. The optimal working dilutions for the monoclonal antibody El3 against IEA (Biosoft) and for the horseradish peroxidase conjugated goat anti-mouse immunoglobulin G (Pasteur Production) were determined in a checkerboard testing on MRC-5 cell monolayers infected with AD169 strain. All antibody incubations were at 37°C for 30 min, separated by three washings in phosphate buffered saline without Ca2+ and Mg2+. Diaminobenzidine tetra hydrochloride/H202 was used as the peroxidase substrate. The entire bottom of each well was examined for the presence of infected cells with an inverted microscope at a lOO-fold magnification and IEA positive nuclei stained brown were counted.
314 TABLE 1 Comparative sensitivities of MRC-5, retinoic acid induced Tera-2, U-373MG, BHK2,, ML, BALB/ c-3T3, Y-l cultures for detection of AD169 CMV strain IEA. AD169 dilution 10-3 lo-4
Cell cultures MRC-5
Tera-2b
U-373MG
BHKz,
ML
BALB/c-3T3
Y-l
117= 12
935 102
29 3
125 14
188 44
11 1
50 6
a Values are expressed as a mean of the number of marked nuclei per well in quadruplicate cultures. b Tera-2 cells were treated with IO-’ M RA for 10 days before infection and during the incubation of infected cells.
Results
The comparative sensitivities of human and non human cell cultures for the detection of immediate early gene expression at 24 h post inoculation with CMV strain AD169 are shown in Table 1. The number of positive nuclei was 2to 3-fold higher in ML cell cultures and IO-fold higher in RA-induced Tera-2 cell cultures than in MRC-5 monolayers. The numbers of positive nuclei were equivalent in BH&i and MRC-5 cells, whereas in U-373MG, Balb/c-3T3 and Y-l cell monolayers, only small numbers of IEA producing cells were detected. The comparative sensitivities of MRC-5, ML and RA-induced Tera-2 cell cultures for detection of CMV in 63 urine samples are shown in Table 2. At 24 h post-infection, 20 (3 1.7%) of the 63 specimens were positive by centrifugation culture. All 20 positive specimens were detected in MRC-5 cells, whereas only 18 (28.5%) were detected in ML or RA-induced Tera-2 cells. Moreover, the number of infected nuclei detected in MRC-5 cells as compared to ML and RA-induced Tera-2 cells was greater (Table 3). Three samples were toxic to the Iibroblast cell monolayer. However, sufficient cells remained in wells to permit interpretation of the results. The major problem with RA-induced Tera-2 cells was toxicity which resulted in deterioration, but not complete destruction, of the monolayer. In contrast, ML cells were less affected by the toxicity present in some clinical specimens than tibroblasts. The sensitivities of MRC-5 and ML TABLE 2 Comparison of 24-well plate cell cultures of MRC-S, ML and differentiated CMV in urine specimens
Tera-2 for detection of
Snecimen numbers
MRC-5
ML
RA-induced
43 17 1 1 1
_a + + + +
+ + -
_ + + -
’ + , positive samples; - , negative samples.
Tera-2
315 TABLE 3 Comparison of centrifugation cultures of MRC-5, ML and RA-induced Tera-2 cells for detection of HCMV in urine specimens. AD169 controls are included. The mean numbers of marked nuclei per well were indicated Specimens AD169 AD169 : :
MRC-5
ML
RA-induced
150 50 106 337 21 157 66
520 280
820 330
::
:: 8 47 54 0 40 30 3
92
2: 63 0 17 26
11 6 178
:, 8
2 1:; ; 9 10 11 12 13 :‘: :; 18 :z
2::: ld; : 21 32 10
Tera-2
4: 4 40
2; 3 142 1 1
23%
3 1
2: 4
;
cell cultures for CMV detection were also compared at 48 h post infection. Twenty-two positive specimens were detected in MRC-5 and only 20 in ML cells. To determine the sensitivity of different passages of ML cells to CMV wild strains, 15 positive urine samples were thawed and inoculated into ML cell monolayers at passages 60 and 110. Twelve were again found positive and the number of stained nuclei was not significantly different in high passage cell culture as compared to low passage cell culture.
Conclusion Rapid CMV culture procedures using human tibroblasts are widely used in clinical laboratories. The centrifugal enhancement of HCMV infectivity is well documented (Mazeron et al., 1984; Gleaves et al., 1984; Alpert et al., 1985; Ho and Plotkin, 1987) and centrifugation of the inoculated cell cultures is a crucial component of the method for rapid diagnosis. The two major disadvantages of human fibroblast cultures are the centrifugal enhancement .of the susceptibility of these cells to the toxic effects of some clinical specimens (Alpert et al., 1985; Paya et al., 1988) and the short duration of their usefulness in the laboratory (generally passages 21 to 36). To overcome these problems, different continuous cell lines, which do express CMV LEA after infection, were
316
assessed for rapid diagnosis. MRC-5 fibroblastic cells were found to be more sensitive for AD169 IEA expression than human U-373MG and non-human (Balb/c-3T3, Y-l) cell lines. Moreover, U-373MG cells were shown to be less sensitive than MRC-5 to CMV isolate IEA expression in a recent report (Boeckh et al., 1991). BHKzl and MRC-5 cells were equivalent in sensitivity. BHKZI, Balb/c-3T3, and Y-l cultures were not investigated further as confluent monolayers were difficult to maintain. ML and RA-induced Tera-2 cells showed a differential tropism for the different kinds of virus. Unexpectedly, they expressed more positive nuclei than MRC-5 cells at 24 h post-infection with AD169 strain that has been extensively adapted to growth in tibroblasts, but less positive nuclei when they were infected by wild strains. ML cells were found to be more sensitive than HF for CMV detection by MacKenzie and McLaren (1989) who reported an even greater sensitivity of high passage cell cultures. ML cells used in this study did not show the expected sensitivity and no change in sensitivity was obtained with high passage cultures. The great toxicity observed in differentiated Tera-2 cells with clinical specimens could explain the lack of sensitivity of these cells. This comparative study indicates that MRC-5 cell cultures are the most sensitive cell culture system tested for rapid CMV detection from clinical specimens. References Alpert, G., Mazeron, MC., Colimon, R. and Plotkin, S. (1985) Rapid detection of human cytomegalovirus in the urine of humans. J. Infect. Dis. 152, 631633. Boeckh, M., Gleaves, C.A., Bindra, R. and Meyers, J.D. (1991) Comparison of MRC-5 and U373MG astrocytoma cells for detection of cytomegalovirus in shell vial centrifugation cultures. Eur. J. Clin. Microbial. Infect. Dis. 10, 569-572. Gleaves, C.A, Smith, T.F., Shuster, E.A. and Pearson, G.R. (1984) Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbial. 19, 917-919. Gijncziil, E., Andrews, P.W. and Plotkin, S.A. (1985) Cytomegalovirus infection of human teratocarcinoma cells in culture. J. Gen. Virol. 66, 509-515. Ho, W. and Plotkin, S.A. (1987) Rapid detection of cytomegalovirus infections by a tissue culturecentrifugation-monoclonal antibody-biotin/avidin immunofluorescence technique. Mol. Cell. Probes. 1, 83-93. Lafemina, R.L. and Hayward, G.S. (1986) Constitutive and retinoic acid-inducible expression of cytomegalovirus immediate-early genes in human teratocarcinoma cells. J. Virol. 58, 434-440. Lafemina, R.L. and Hayward, G.S. (1988) Differences in cell type-specific blocks to immediate early gene expression and DNA replication in human, simian and murine cytomegalovirus. J. Gen. Virol. 69, 355-374. MacKenzie, D. and McLaren, L.C. (1989) Increased sensitivity for rapid detection of cytomegalovirus by shell vial centrifugation assay using mink lung cell cultures. J. Virol. Methods. 26, 183-188. Mazeron, M.C., Colimon, R., Roseto, A. and Perol, Y. (1984) Detection of cytomegaloviremia using monoclonal antibodies. Dev. Biol. Standard 57, 287-291. Paya, C.V., Wold, A.D., Ilstrup, D.M. and Smith, T.F. (1988) Evaluation of number of shell vial
317 cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection. J. Clin. Microbial. 26, 198-200. Tanaka, J., Ogura, T., Kamiya, S., Yoshie, T., Yabuki, Y. and Hatano, M. (1984) Dexamethasone enhances human cytomegalovirus replication in human epithelial cell cultures. Virol. 136, 448452. Tanaka, J., Kamiya, S., Ogura, T., Sato, H. and Hatano, M. (1985) Effect of dimethylsulfoxide on interaction of human cytomegalovirus with host cell: conversion of a non-productive state of cell to a productive state for virus replication. Virology 146, 165-176. Smiley, M.L., Mar, E.C. and Huang, E.S. (1988) Cytomegalovirus infection and viral-induced transformation of human endothehal cells. J. Med. Virol. 25, 213226. Smith, J. (1986) Human cytomegalovirus: demonstration of permissive epithelial cells and non permissive fibroblastic cells in a survey of human cell lines. J. Virol. 60, 583-588.
1992 Elsevier Science Publishers
311
B.V. / All rights reserved / 0166-0934/92/$05.00
VIRMET 01390
Comparison of MRC-5 and continuous cell lines for detection of cytomegalovirus in centrifugation cultures Marie-Christine Mazeron, Badia Benjelloun, Claire Bertrand, Jean-Luc Pons and Yvonne PQol Service de BactPriologie-Virologie.
H6pital Saint-Louis,
UniversitP Paris VII, Paris (France)
(Accepted 5 May 1992)
Summary
Continuous cell lines were assessed for use for rapid human cytomegalovirus (HCMV) detection procedures combining tissue culture, centrifugation, and immediate early antigen (IEA) immunostaining. Human cells (MRC-5 embryonic fibroblasts, U373MG astrocytoma cells, differentiated teratocarcinoma (Tera-2) cells), murine cells (BALB/c-3T3 and Y-l cells), BHK:!r hamster cells, and mink lung (ML) cells were first inoculated with HCMV laboratory strain. IEA synthezising cells were detected by immunoperoxidase assay using a monoclonal antibody. ML cells and differentiated Tera-2 cells exhibited more positive cells than MRC-5 cells. BHK 21, and MRC-5 cells were equivalent in sensitivity whereas U-373MG, BALB/c-3T3, and Y-l cells had only reduced IEA positive cells. When 63 urine specimens were inoculated onto MRC-5, ML and differentiated Tera-2 cells, 20 (31.7%) were positive in MRC-5 cells versus 18 (28.5%) in ML or Tera-2 cells. Moreover, greater numbers of infected cells were detected in MRC-5 cells than in these two cell lines. MRC-5 cells were superior for detection of HCMV in clinical samples by centrifugation cultures. Cytomegalovirus (human); Centrifugation culture; Cell lines, continuous; Fibroblast (human); Immediate early antigen
Correspondence to: M.C. Mazeron, Service de Bactkiologie-Virologie, Ambroise Park, 7’5475,Paris Ctdex 10, France.
HBpital LariboisDre, 2, rue
312
Introduction Human cytomegalovirus (HCMV) is highly restricted in its host range in cell cultures. Efficient replication and release of high titers of progeny virions are obtained only in human diploid tibroblasts (HF). Other human cell types tested, such as epithelial cells (Smith, 1986; Tanaka et al., 1984), endothelial cells (Smiley et al., 1988), rabdomyosarcoma cells (Tanaka et al., 1985) are moderately permissive. Human teratocarcinoma stem cells are non-permissive for HCMV but become permissive after being induced to differentiate by treatment with retinoic acid (RA) (Goncziil et al., 1984; LaFemina and Hayward, 1986). In non-permissive cells, failure in progression of the lytic cycle occurs at any of the steps between adsorption and virion production (LaFemina and Hayward, 1988). HCMV immediate early antigen (IEA) expression does occur in some non-permissive xenogeneic cell lines. HCMV isolation procedures are usually carried out using HF. Rapid methods use a combination of low-speed centrifugation of the clinical specimens onto cell monolayers and detection of IEA in infected cells (Mazeron et al., 1984; Gleaves et al., 1984). As these methods do not necessitate a complete viral replication cycle, continuous cell lines which allow expression of IEA following infection by HCMV strains could be used for diagnosis. These would offer advantages for the clinical laboratory compared to HF, which are generally useful only over a limited number of passages. Mink lung (ML) cell cultures were found to be more sensitive than HF for detection of HCMV strains (MacKenzie and McLaren, 1989). In this study, human and non-human cell cultures were compared for the degree of sensitivity to IEA expression of HCMV reference and wild strains.
Materials and Methods Cell cultures The sources of the cell lines used were as follows: MRC-5 human embryonic lung tibroblasts, passages 28 to 36, (Bio-Merieux); I-I-373MG human astrocytoma cells, (J-L Virelizier, Institut Pasteur, Paris); Tera-2 human teratocarcinoma stem cells, BALB/c-3T3 established murine cells, BHK*i(C-13) established hamster kidney cells and ML cells (G Peries, Paris, Inserm U-107); Y-l murine adrenal tumor cells (American Type Culture Collection, ATCC CCL 79). Y-l cells were cultured in Ham’s FlO medium containing 15% horse serum and 2.5% fetal bovine serum and the other cells in Eagle’s Minimal Essential Medium (MEM) with 10% fetal bovine serum. Differentiation of Tera-2 cells was achieved by subculture in the same medium containing RA (Aldrich) at a concentration of 10v5 M for 10 days. Cell monolayers grown in 24-well plates were used for HCMV isolation. The number of cells seeded in each well was adjusted to obtain subconfluent
313
monolayers within 48 to 72 h for MRC-$24
and 48 h for the other cell types.
HCMV reference strain
The AD169 strain of HCMV (ATTC VR-538) was propagated in MRC-5 cell cultures. The cells were inoculated at a multiplicity of infection of one. When cytopathic effects reached 90%, supernatants were saved, clarified by low speed centrifugation and retained as virus stocks. These stocks were dispensed in l-ml aliquots and stored at -80°C. Immediately before use, a stock aliquot was thawed and diluted serially IO-fold. Clinical specimens and processing
Routine urine specimens submitted for HCMV cultures were obtained from marrow and renal transplant recipients and patients with the acquired immunodeficiency syndrome. The specimens were collected in an equal volume of transport medium (MEM containing 10% fetal bovine serum, sorbitol and antibiotics) and kept on ice during transportation to the virology laboratory. For processing specimens, 5 ml of each urine sample was filtered through a 0.45~pm pore size filter (Sartorius). After inoculation, all specimens were stored at -80°C. Rapid detection of CMV Inoculation and incubation of specimens. Immediately before inoculation, the culture medium was decanted from the cell monolayers in the plates. 0.1 ml of diluted viral stock was added to each of four wells and 0.5 ml of processed urine samples were inoculated into each of two wells. The supernatant volume in each well was made up to 1 ml with maintenance medium. Inoculated 24-well plates were centrifuged at 2900 x g at 36°C for 45 min. Inocula were then removed and replaced by fresh medium. The cells were incubated at 37°C in 5% COZ atmosphere for 24 or 48 h. Fixation and staining procedures. Cells were fixed in a mixture of cold acetone and distilled water (9: 1, v/v) at -20°C for 10 min. The optimal working dilutions for the monoclonal antibody El3 against IEA (Biosoft) and for the horseradish peroxidase conjugated goat anti-mouse immunoglobulin G (Pasteur Production) were determined in a checkerboard testing on MRC-5 cell monolayers infected with AD169 strain. All antibody incubations were at 37°C for 30 min, separated by three washings in phosphate buffered saline without Ca2+ and Mg2+. Diaminobenzidine tetra hydrochloride/H202 was used as the peroxidase substrate. The entire bottom of each well was examined for the presence of infected cells with an inverted microscope at a lOO-fold magnification and IEA positive nuclei stained brown were counted.
314 TABLE 1 Comparative sensitivities of MRC-5, retinoic acid induced Tera-2, U-373MG, BHK2,, ML, BALB/ c-3T3, Y-l cultures for detection of AD169 CMV strain IEA. AD169 dilution 10-3 lo-4
Cell cultures MRC-5
Tera-2b
U-373MG
BHKz,
ML
BALB/c-3T3
Y-l
117= 12
935 102
29 3
125 14
188 44
11 1
50 6
a Values are expressed as a mean of the number of marked nuclei per well in quadruplicate cultures. b Tera-2 cells were treated with IO-’ M RA for 10 days before infection and during the incubation of infected cells.
Results
The comparative sensitivities of human and non human cell cultures for the detection of immediate early gene expression at 24 h post inoculation with CMV strain AD169 are shown in Table 1. The number of positive nuclei was 2to 3-fold higher in ML cell cultures and IO-fold higher in RA-induced Tera-2 cell cultures than in MRC-5 monolayers. The numbers of positive nuclei were equivalent in BH&i and MRC-5 cells, whereas in U-373MG, Balb/c-3T3 and Y-l cell monolayers, only small numbers of IEA producing cells were detected. The comparative sensitivities of MRC-5, ML and RA-induced Tera-2 cell cultures for detection of CMV in 63 urine samples are shown in Table 2. At 24 h post-infection, 20 (3 1.7%) of the 63 specimens were positive by centrifugation culture. All 20 positive specimens were detected in MRC-5 cells, whereas only 18 (28.5%) were detected in ML or RA-induced Tera-2 cells. Moreover, the number of infected nuclei detected in MRC-5 cells as compared to ML and RA-induced Tera-2 cells was greater (Table 3). Three samples were toxic to the Iibroblast cell monolayer. However, sufficient cells remained in wells to permit interpretation of the results. The major problem with RA-induced Tera-2 cells was toxicity which resulted in deterioration, but not complete destruction, of the monolayer. In contrast, ML cells were less affected by the toxicity present in some clinical specimens than tibroblasts. The sensitivities of MRC-5 and ML TABLE 2 Comparison of 24-well plate cell cultures of MRC-S, ML and differentiated CMV in urine specimens
Tera-2 for detection of
Snecimen numbers
MRC-5
ML
RA-induced
43 17 1 1 1
_a + + + +
+ + -
_ + + -
’ + , positive samples; - , negative samples.
Tera-2
315 TABLE 3 Comparison of centrifugation cultures of MRC-5, ML and RA-induced Tera-2 cells for detection of HCMV in urine specimens. AD169 controls are included. The mean numbers of marked nuclei per well were indicated Specimens AD169 AD169 : :
MRC-5
ML
RA-induced
150 50 106 337 21 157 66
520 280
820 330
::
:: 8 47 54 0 40 30 3
92
2: 63 0 17 26
11 6 178
:, 8
2 1:; ; 9 10 11 12 13 :‘: :; 18 :z
2::: ld; : 21 32 10
Tera-2
4: 4 40
2; 3 142 1 1
23%
3 1
2: 4
;
cell cultures for CMV detection were also compared at 48 h post infection. Twenty-two positive specimens were detected in MRC-5 and only 20 in ML cells. To determine the sensitivity of different passages of ML cells to CMV wild strains, 15 positive urine samples were thawed and inoculated into ML cell monolayers at passages 60 and 110. Twelve were again found positive and the number of stained nuclei was not significantly different in high passage cell culture as compared to low passage cell culture.
Conclusion Rapid CMV culture procedures using human tibroblasts are widely used in clinical laboratories. The centrifugal enhancement of HCMV infectivity is well documented (Mazeron et al., 1984; Gleaves et al., 1984; Alpert et al., 1985; Ho and Plotkin, 1987) and centrifugation of the inoculated cell cultures is a crucial component of the method for rapid diagnosis. The two major disadvantages of human fibroblast cultures are the centrifugal enhancement .of the susceptibility of these cells to the toxic effects of some clinical specimens (Alpert et al., 1985; Paya et al., 1988) and the short duration of their usefulness in the laboratory (generally passages 21 to 36). To overcome these problems, different continuous cell lines, which do express CMV LEA after infection, were
316
assessed for rapid diagnosis. MRC-5 fibroblastic cells were found to be more sensitive for AD169 IEA expression than human U-373MG and non-human (Balb/c-3T3, Y-l) cell lines. Moreover, U-373MG cells were shown to be less sensitive than MRC-5 to CMV isolate IEA expression in a recent report (Boeckh et al., 1991). BHKzl and MRC-5 cells were equivalent in sensitivity. BHKZI, Balb/c-3T3, and Y-l cultures were not investigated further as confluent monolayers were difficult to maintain. ML and RA-induced Tera-2 cells showed a differential tropism for the different kinds of virus. Unexpectedly, they expressed more positive nuclei than MRC-5 cells at 24 h post-infection with AD169 strain that has been extensively adapted to growth in tibroblasts, but less positive nuclei when they were infected by wild strains. ML cells were found to be more sensitive than HF for CMV detection by MacKenzie and McLaren (1989) who reported an even greater sensitivity of high passage cell cultures. ML cells used in this study did not show the expected sensitivity and no change in sensitivity was obtained with high passage cultures. The great toxicity observed in differentiated Tera-2 cells with clinical specimens could explain the lack of sensitivity of these cells. This comparative study indicates that MRC-5 cell cultures are the most sensitive cell culture system tested for rapid CMV detection from clinical specimens. References Alpert, G., Mazeron, MC., Colimon, R. and Plotkin, S. (1985) Rapid detection of human cytomegalovirus in the urine of humans. J. Infect. Dis. 152, 631633. Boeckh, M., Gleaves, C.A., Bindra, R. and Meyers, J.D. (1991) Comparison of MRC-5 and U373MG astrocytoma cells for detection of cytomegalovirus in shell vial centrifugation cultures. Eur. J. Clin. Microbial. Infect. Dis. 10, 569-572. Gleaves, C.A, Smith, T.F., Shuster, E.A. and Pearson, G.R. (1984) Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbial. 19, 917-919. Gijncziil, E., Andrews, P.W. and Plotkin, S.A. (1985) Cytomegalovirus infection of human teratocarcinoma cells in culture. J. Gen. Virol. 66, 509-515. Ho, W. and Plotkin, S.A. (1987) Rapid detection of cytomegalovirus infections by a tissue culturecentrifugation-monoclonal antibody-biotin/avidin immunofluorescence technique. Mol. Cell. Probes. 1, 83-93. Lafemina, R.L. and Hayward, G.S. (1986) Constitutive and retinoic acid-inducible expression of cytomegalovirus immediate-early genes in human teratocarcinoma cells. J. Virol. 58, 434-440. Lafemina, R.L. and Hayward, G.S. (1988) Differences in cell type-specific blocks to immediate early gene expression and DNA replication in human, simian and murine cytomegalovirus. J. Gen. Virol. 69, 355-374. MacKenzie, D. and McLaren, L.C. (1989) Increased sensitivity for rapid detection of cytomegalovirus by shell vial centrifugation assay using mink lung cell cultures. J. Virol. Methods. 26, 183-188. Mazeron, M.C., Colimon, R., Roseto, A. and Perol, Y. (1984) Detection of cytomegaloviremia using monoclonal antibodies. Dev. Biol. Standard 57, 287-291. Paya, C.V., Wold, A.D., Ilstrup, D.M. and Smith, T.F. (1988) Evaluation of number of shell vial
317 cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection. J. Clin. Microbial. 26, 198-200. Tanaka, J., Ogura, T., Kamiya, S., Yoshie, T., Yabuki, Y. and Hatano, M. (1984) Dexamethasone enhances human cytomegalovirus replication in human epithelial cell cultures. Virol. 136, 448452. Tanaka, J., Kamiya, S., Ogura, T., Sato, H. and Hatano, M. (1985) Effect of dimethylsulfoxide on interaction of human cytomegalovirus with host cell: conversion of a non-productive state of cell to a productive state for virus replication. Virology 146, 165-176. Smiley, M.L., Mar, E.C. and Huang, E.S. (1988) Cytomegalovirus infection and viral-induced transformation of human endothehal cells. J. Med. Virol. 25, 213226. Smith, J. (1986) Human cytomegalovirus: demonstration of permissive epithelial cells and non permissive fibroblastic cells in a survey of human cell lines. J. Virol. 60, 583-588.
