Rapid detection of active cytomegalovirus infection by in situ polymerase chain reaction on MRC5 cells inoculated with blood specimens

Journal

of Virological

Methods

49 (1994) 59 66

Rapid detection of active cytomegalovirus infection by in situ polymerase chain reaction on MRC5 cells inoculated with blood specimens D. Bettinger*,

C. Mougin,

Lahortrtoire c/e [email protected]. L:. Suint-Jucques, Accepted

M. Lab

2, place Saint-Jucyue.~

IO February

, 25000BCWIFO~~,Frww

1994

Abstract

An in situ polymerase

chain reaction was developed to amplify immediate early genes of human cytomegalovirus in cells cultured in a 96 well plate and infected with leukocytes. The technical parameters enabling optima1 detection of the DNA sequences were defined. The key to this method is the fixation of cells, which facilitates the access of the PCR mixture into the cell nuclei and preserves cell morphology. Such a technique could have wide application for the detection and identification of other infectious viruses in cultured cells very early after inoculation of clinical samples. Ke_r words:

Human cytomegalovirus;

in situ PCR; MRC5 tibroblasts:

leukocytes

I. Introduction

Active human cytomegalovirus (CMV) infection causes a variety of syndromes in immunocompromised patients (Alford and Britt, 1993). An accurate and rapid diagnosis of infectious CMV is therefore of vital importance, since effective anti-CMV agents are now available to eradicate CMV infection (Collaborative DHPG Treatment Study Group, 1986; Goodrich et al., 199l).Diagnosis of CMV infection is based on serological data (De Ory et al., 1988; Revello et al., 1991), and/or the detection of the virus from any site. Direct detection of CMV can be achieved by demonstration of cellular lesions (Rasing et al., 1990; Theise et al., 1991), the presence of antigens (Bein et al., 1991; Gerna et al., 1992; Van Den Berg et al., 1991) or *Corresponding

author.

l-ax: + 33 81665744.

0166.0934/94:$07.00 I( 1994 Elsevier Science B.V. All rights rcscrved SSDI 0166-0934(94)00032-C

nucleic acids (Dankner et al., 1990; Gleaves et al., 1989; Hsia et al., 1989; Jiwa et al., 1989; Musiani et al., 1990; Olive et al., 1989; Shibata et al., 1988). However, the presence of antigens or viral DNA does not always correlate with viremia or clinical symptoms (Gerna et al., 1991) whereas virus isolation after inoculation of monolayers of human fibroblasts, appears to be the most reliable indicator of CMV infection (Geoffrey et al., 1987: McClintock et al., 1989: Miller et al., 1989: Sorbello et al.. 1988). In a previous paper (Mougin et al.. 1991), we described an in situ hybridization (ISH) procedure to detect nucleic acid replication in human embryonic tibroblast cells (MRC5) cultivated in a 96 microwell plate and inoculated with peripheral blood leukocytes (PBL). However, the ISH gave an optimal signal 72 h after infection. To detect CMV DNA sequences within 24 h p.i., it was decided to develop an in situ polymerase chain reaction (PCR) and define conditions for optimal detection of the CMV amplified DNA sequences.

2. Materials and methods The method relied on the use of immediate early (IE) CMV specific primers and dNTP labeled with biotin. The PCR products were detected directly in the cells by alkaline phosphatase-conjugated avidin and the corresponding substrate. In this method, the optimal detection of in situ PCR signal is closely dependent on the fixation and permeability of cells. To fix and ensure permeability of the cells, several treatments were tested: (a) ice cold mixture of ethanol/acetone or acetone:’ water (9O:lO (v;‘v) for 20 min; (b) 2% neutral buffered formalin (2% formalin in 0.1 M sodium phosphate buffer pH 7.2) for 20 min, 60 min or 16 h; (c) 2% formalin for 20 min at room temperature followed by ethanol/acetone solution for 20 min at 4 C. Fixation with acetone/water proved inefficient. After a I6 h fixation with 2% formalin. cells demonstrated a positive signal but the background was intense. Fixation with ethanol;‘acetone or 2% formalin for 20 min or 60 min provided a faint signal without any background. The combination of _7% formalin and ethanol/acetone fixation for 20 min each yielded a maximal CMV DNA detection of PCR product. Since the quality of the stainings and the cellular morphology were poorly preserved by incubation of cells in a proteinase K solution, in situ PCR was carried out without any digestion. The best results were obtained as follows: human embryonic fibroblast cells (MRCS) were plated (1.5 x IO”;well) in RPM1 1640 medium supplemented with 5% fetal calf serum (FCS) into flat bottomed wells of a 96-well microtiter plate. After incubation for 48 h at 37’C in an atmosphere containing 5% CO?. the medium was removed and the monolayers were overlaid with 0.2 ml of virus sample as follows. Isolation of CMV from blood was carried out by using buffy coat cells, which were separated from heparinized peripheral blood. The cells isolated from 7 ml of blood were suspended in 1 ml of RPM1 medium containing 0.1% dexamethasone and 1% DMSO, and the monolayers of MRCS were inoculated with 0.2 ml of the cells. The cultures were centrifuged for 1 hour at low speed (1200 t-pm) at 30 C to incrcasc virus infectivity. The resulting supernatant fluid was then removed and replaced

D. Brrtingrr et al.:Jottmai

o_fVimlogical Methods 49 (1994) 59d6

61

with 0.2 ml RPM1 supplemented with 10% FCS. The monolayers of MRCS were incubated at 37°C in an atmosphere containing 5% COz. The overlay medium was removed 16 h after infection, and the cells were washed with phosphate-buffered saline (PBS) and fixed with 2% formalin and ethanol/acetone. The primers for amplification of CMV sequences were derived from structural analysis of the major immediate early gene of HCMV described by Stenberg et al. (1984). The sequences used were those described by Shibata et al. (1988) (5’ to 3’) CCA CCC GTG GTG CCA GCT CC (IEl, upstream primer) and CCC GCT CCT CCT GAG CAC CC (IE2, downstream primer). The primers were synthetized by Genset (Paris, France) and used after purification. After removing carefully the fixation mixture, PCR was carried out in each well in a total volume of 40 ~1 (reaction buffer: 20 mM Tris (pH X.5), 50 mM KCl, 2 mM MgCIz) containing 80 ng of each primer, 8 nmol of each dNTP (including 5 nmol of biotin-14-dATP (Bethesda Research Laboratories)). Twenty ~1 of the amplifying solution were placed over the fixed cells. After an initial gentle centrifugation (1200 rpm for 2 min) to favour an optimal repartition of the PCR reagent in each well, the target DNA sequences were denatured by heating the microtiter plate on the block of a Thermal Cycler (Hybaid, Schleicher and Schuell) for 4 min at 96°C. Then, one unit Tuq polymerase (AmpliTaq DNA polymerase, Perkin Elmer Corporation, USA) diluted in the other 20 ~1 of PCR mixture was added in each well. To avoid evaporation of the amplifying solution, the space between each well was filled with distilled water. The microwell plate was covered with a sticky paper sheet. After a second centrifugation (1200 rpm for 2 min), 25 cycles were completed in the Thermal Cycler, using the following steps: denaturation at 96°C for 1 min and annealing/ extension at 55°C for 1 min. After washing with PBS, detection of biotin incorporated into PCR products was carried out with 50 $ of alkaline phosphatase-conjugated avidin (Dako) at a I:200 dilution in 0.02 M sodium phosphate monobasic, 0.08 M sodium phosphate dibasic, 0.1 M NaCl, 5% bovine serum albumin (BSA) and 1 pi/ml Triton X-100, for 45 min at 37°C. The wells were then rinsed in PBS. Finally, 50 ~1 of the chromogen nitroblue tetrazolium (NBT) (4.4 pi/ml) with Sbromo-4-chloro-3-indolyl-phosphate (BCIP) (3.2 PI/ml) diluted in 0.1 M Tris (pH 9.6), 0.1 M NaCl, 50 mM MgCl2, yielded a purple-blue precipitate, located in the fibroblast nuclei. The controls of specificity were set up as described above by using non-specific primers, such as consensus human papillomavirus primers, by omitting the Taq polymerase in fibroblasts inoculated with PBL and by running in situ PCR on ~broblasts infected or not with the CMV strain AD 169 diluted l:lO, 1: 100, 1: 1000 in RPM1 medium with 0.1% dexamethasone and 1% DMSO..

3. Results and discussion With the in situ PCR, we were able to demonstrate for the first time that infectious CMV DNA sequences can be detected specifically in cell culture using biotinylated deoxynucleotide triphosphate. Results exactly matched previously validated immediate early gene detection by in situ hybridization (Mougin et al., 1991). In

Fig. 1. Detection of CMV nucleic acid sequences in MRC5 libroblasts (A) In situ polymerase chain reaction performed on MRC5 cells 16 h after infection by leukocytes (X 75). (B) Higher magnification of (A) showing a clear intcnsc staining ( x 480). (C F) Controls of specdiaty of the in situ PCR assay. (C) Absence of slgnal in fibroblasts inoculated with PBL and treated upon addition of the PCR mixture without 7’uq polymcrasc ( x 75). (D) Classical in situ hybridization performed on MRC5 cells 72 h after infection by leukocytes, as described by Mougin et al. (1991) (x 480). (E) In situ PCR test in uninfected MRC5 cells, there is no labcling (x 300). (F) In tibroblasts infected with the CMV strain AD 169 at a dilution I .50, CMV DNA amplified sequences are concentrated within cell nuclei ( x 480).

the present experiment, the specific synthesis of the CMV DNA was clearly detectable with the strain AD 169 at a dilution 1: 10 or I: 100, while the signal decreased with the strain diluted 1:lOOO. The efficiency of amplification of CMV DNA by PCR thus appeared to enhance the signal detection at least IO-fold, in comparison with the in situ hybridization (Mougin et al., 1991). The steps of the technique described now were reproductible and consistently provided a nuclear staining

(Fig. 1). The labeling occasionally extended to the cytoplasm. The intensity of the in situ PCR signal also varied widely from cell to cell, indicating that CMV infection is highly cell-associated. Among the methods developed for the detection of CMV, PCR is the most powerful (Boland et al., 1992; Brytting et al., 1991; Buffone et al., 1990; Einsele et al., 1991; Lorino et al., 1993). But in no case was the actual infectivity of the virus confirmed thus (Delgado et al., 1992; Gerdes et al., 1993; Stanier et al., 1989), except when PCR was carried out after reverse transcription of late mRNA (Bitsch et al., 1993; Gozlan et al., 1992). The rapidity plus the qualitative and quantitative nature of this PCR experiment without extracting DNA from cell lysates suggests that prompt detection of infectious CMV can be obtained by this in situ DNA amplification procedure. Detection of a viremia by antibodies or by CMV probes in infected cultured cells appeared a useful tool for confirming CMV infection. Earlier detection of viremia by in situ PCR would permit an earlier antiviral therapy and may be more successful in CMV-infected immunosuppressed patients. Some procedures for in situ PCR in fixed tissues or cells have already been described (Nuovo et al., 1991), but have never been reported on cultured cells, or on infected cells to study viral replication in vitro. The adjustment of the technique showed the critical influence of fixation and digestion in order to allow the amplification reagents to reach the DNA target in the cells. Like Nuovo et al. (1993), we have noted that a successful in situ PCR is hindered by fixation with acetone. Thus, acetone will not prevent migration of the amplified product out of the nucleus, probably because proteins and, perhaps, nucleic acids are not cross-linked (Nuovo et al., 1993). By contrast, formalin fixation for 16 h clearly allowed to create a physical barrier preventing migration of the PCR product, because such fixative polymerizes proteins and can cross-link nucleic acids (Nuovo, 1989). But the strong background observed in our cultured cells led to reduction of the fixation period, which apparently can inhibit the entry of one key PCR reagent. Thus, ethanol appears to be a necessary step to enhance sensitivity of the present method. Finally, under the described experimental conditions, there is a minimal migration of the 159 bp amplified product from its original site, whereas theoretically small fragments (~450 bp) should have been melnbrane-permeant and should have diffused from the nuclei. We are currently studying the detection of CMV DNA by PCR in parallel with antigen detection with the monoclonal antibody antiprotein kinase 65-68, the lC3 (Biosoft, Paris) which recognizes the internal matrix structural phosphoprotein present in the nuclei of infected cells as early as the second hour post-infection (Gerna et al., 1992). This comparison was carried out to determine if in situ PCR consistently detects CMV viremia earlier than immunocytochemical technique. To date, cells infected with serial dilution of CMV strain AD 169 and incubated with the mAb have displayed a much lower number of stained inclusions at both dilutions I:10 and 1:lOO and no inclusion at the dilution of 1:lOOO. Moreover, the use of in situ PCR to study the presence of virus in cell population or tissue has been somewhat limited by the lack of automation, although the initial appIication of this in situ PCR has stressed the importance of this new technology.

The combined sensitivity, specificity, rapidity and cost effectiveness make this in situ PCR assay a more powerful alternative to in situ hybridization for the diagnosis of active CMV infection. This test carried out on cells cultured in 96-microtitration plates can be applied for routine diagnosis to screen a large number of clinical samples. It has therefore the potential to replace the classical. more laborious PCR in conjunction with the demonstration of a virus active replication. Suitable controls must however be incorporated in each experiment to exclude false negative results. e.g.. due to PCR inhibitors or false positive results and sample contaminations.

Acknowledgements

We thank Laurence Madoz and Jean Michel Jounet for excellent technical assistance and Manon Lab for many helpful English suggestions. This work was supported from funds of Groupe de Recherche en Virologie de Franche-Comtk.

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