Rapid detection of cytomegalovirus using immune scanning electron microscopy

Journal of Virological Me&o&

16 (1987) 55-64

Elsevier JVM 00580

Rapid detection of cytomegalovirus using immune scanning electron microscopy J. Anderssonl,

R. Nybom’, P. Larsson2, U. Anderssonl, A. Ehrnst4

S. Britton3 and

‘Department of Immunology, Stockholm University, Sweden; ‘Department of Clinical Immunology, Karolinska Hospital, Stockholm. Sweden; 3Roslagstull Hospital, Stockholm, Sweden; 4Department of Virology, Stockholm County Council, Central Microbiological Laboratory, Sweden

(Accepted 6 January 1987)

Summary Monoclonal antibodies specific for human cytomegalovirus were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex bead surface. No such binding of virus particles were detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids. Cytomegalovirus; Virus diagnosis; Scanning electron microscopy -_ --------__._~

_“.._

Introduction Human cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in endogenously or iatrogenically immunosuppressed patients (Macher et al., 1983). The recovery of CMV from urine or oropharyngeal secretions is Correspondence address: J. Andersson, Department University, 106 91 Stockholm, Sweden.

of Immunology, Biology Building F, Stockholm

0166-0934/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

56

common but it does not distinguish between a virus-carrier state and a virus-induced disease (Stagano et al., 1975). CMV infection in peripheral blood is, at present, considered to be a nonpermissive cell-associated virus process (Rice et al., 1984). However, the isolation of CMV from peripheral blood or blood leukocyte cultures is thought to indicate a clinical infection (Fiala et al., 1975). The methods normally used to detect cytomega~oviremia have been the isolation of CMV from buffy coat preparations seeded on human embryonic fibroblasts (Weller et al., 1957), which takes 10-50 days, or the immunofluorescent staining of infected cells (Griffith et al., 1984). Recently, a new method for CMV genome detection by DNA hybridization has been developed (Spector et al., 1984). However, it does not necessarily correlate to a viremic state, since a small number of DNA hybridizationpositive cells can also be found in blood lymphocytes in the virus-carrier state (Schrier et al., 1985). A new filtration method was developed in an attempt to determine whether extracellular CMV particles could be directly identified in serum during the viremic phase. Supernatants from CMV-positive tissue cultures were inoculated with serum and then filtrated through smooth membranes, Diluted materials from such cultures were accumulated on polycarbonate filters of defined porosity measuring 0.2 j.r.rnand analyzed in a scanning electron microscope (SEM), Homogeneous round particles of roughly 200- and lOO-nm size were detected. The filters were then treated with 15 mM EDTA and a ‘grid’ was inverted on the surface, which ahowed visualization by transmission electron microscopy (T-EM) of ZOO-nm-sited enveloped viral particles as well as of 100-nm-sized nucleocapsides. Thus, it was possible to enrich virus particles on membranes by filtration without creating protein accumulation to such an extent that it would cover all particles and make visualization by SEM impossible. However, the objectives of this study were not only to investigate if this technique could be useful for detection of extracellular CMV particles in samples from patients with acute CMV infections, but also to try to develop an immunological method, by use of monoclonal antibodies (MAbs) specific for CMV antigens, for identi~catio~ of the accumulated particles. The specificity and the sensitivity of this combined approach for CMV detection was then compared with conventional virus isolation by tissue culture.

Materials and Methods Cli~ic~l specimef2.s

Serum and urine samples were obtained from bone marrow- or kidney allografttransplanted patients from the Department of Transplantation, Huddinge Hospital, in a period beginning 3 wk after transplantation and ending at the latest 14 wk after allografting.

57

Control virus from clinical samples

Saliva were obtained from patients with acute infectious mononucleosis, and positive Epstein-Barr virus (EBV) isolation by tissue culture. Urine and serum were obtained from healthy CMV-seropositive and -seronegative individuals. Vesicle contents were aspirated from patients with varicella, herpes simplex type 1 and type 2 vesicles. Virus tissue culture isolates

Supernatants centrifuged (1200 x g, 5 min) from various positive virus isolates, produced by tissue cultures, were used as negative and positive controls. The following virus isolates were used: herpes simplex type 1 and type 2, varicella, Epstein-Barr virus (B95-8), CMV (AD i69), adenovirus, influenza A and B, parainfluenza type 3 and HIV (H9). All types of virus were grown at the Department of Virology, Stockholm County Council, Central Microbiological Laboratory. CMV-antibody

test

All the patients were tested for CMV-specific antibodies by an enzyme-linked immunosorbent assay (ELISA) (Sundqvist et al., 1981). ~soiatio~ of CMV in tissue culture

Virus cultures were performed at the National Bacteriological Laboratory, Stockholm, Sweden. Buffy coat and urine were obtained from transplanted patients, upon clinical suspicion of infection, and cultured on human embryonic lung fibroblasts for 8 wk. The isolates were diagnosed as CMV by immunofluorescence or by ELISA (Sundqvist et al., 1981). Antibody-labelled

microsp~eres

Carboxylated coumarin containing latex particles (3.92 pm, Polyscience Inc., USA) were washed 3 times (10000 x g, 3 min) with coupling buffer (3 mM NaH2P0,, pH 6.3). Approx. 5 x 10s particles in 500 p_lwere then mixed with an equal volume of EDC [l-ethyl-3-(3-dimethy aminopropyl)carbodiimide, (Bio-Rad Inc., USA)] at 10 mg/ml in coupling buffer. The particles were subsequently incubated under continuous vigorous agitation for 1 h at room temperature and then washed with deionized water. One ml of affinity-puri~ed rabbit anti-mouse Fcgamma (MIAB, Knivsta, Uppsala, Sweden), at 1 mgimf in coupling buffer, was then added to the particle pellet and this was followed by a brief (ultra)sonication for 30 s. Covalent binding of the rabbit antibodies was allowed to continue overnight at 4°C under continuous agitation. After coupling, the particles were washed 3 times with 50 mM NaH,P,, 0.15 M NaCl, 2% bovine serum albumin, 0.1% Tween 20, and 0.1% NaN, at pH 7.5, suspended in 5 ml of the same buffer, and finally

58

(ultra)sonicated as above. The 2 anti-CMV MAbs, CH 16-20 and CH 12 (Pereira et al., 1982), were added (25 ~1 each) to the particle pellet and the beads were washed after a l-h incubation (37°C) 4 times in 50 mM NaH,PO,, 0.15 M NaCl, 2% bovine serum albumin, 0.1% Tween 20, and 0.1 NaN, at pH 7.5, and finally suspended in 5 ml of the same buffer. Immune scanning electron microscopy Ten thousand anti-CMV-coated beads were incubated under continuous agitation in 100-l.iJ serum or tissue culture sample for 1 h at 37°C. Urine was used in a volume of 200 ~1. The samples were then diluted 1: 10 to 1:lOO in PBS before the microspheres were collected and washed 3 times in PBS on the surface of a polycarbonate 0.2- or 0.6q.m filter (Nucleopore, Inc.), which was fitted airtight to a current leading device (J.S. Medical, Huskvarna, Sweden). The filter was dried in room temperature, coated with 40-A thick layer of ionized gold and analyzed in the SEM (Philips high resolution SEM 515). Five hundred to 1000 beads were examined in each experiment.

Results All the filtration assessments were done prospectively before the results of virus isolation by tissure culture became available. In 2 initial experiments, serum and urine samples were filtrated from 2 transplanted’patients with positive CMV isolation in buffy coat and urine, respectively. Approx. 200-pm-sized particles were amassed and identified on the filter surface of a 0.2~p_rn polycarbonate filter. These were also visualized in the TEM, when transferred to grids after 15-mM EDTA treatment of the filter surface. In order TABLE

1

Comparison identification

between immune scanning in clinical samples.

filtration

test and virus isolation

by tissue culture’

for CMV

No. of patients

Type of material

Type of virus isolated

Binding to MAb-labelled beads*

25 15 20 25 8 10 2

Urine Serum’ Urine Serum3 Saliva Vesicles Vesicles

CMV CMV Negative Negative EBV Herpes simplex Varicella

23125 14115 O/20 0125 018 O/10 o/2

‘CMV isolation was performed from buffy coat (see Methods). ‘Beads (3.92 pm) were labelled with 2 MAbs specific for CMV. At least 500 beads were examined in the SEM. %MV-antibody analysis performed according to Methods. Fifteen of the patients were CMV-seropositive, 10 were CMV-seronegative.

Fig. 1. SEM micrograph (magnification 1.15 X 10’) illustrating human CMV-forming rosettes around antibody-labelled 3.92~pm microspheres incubated in serum from a patient with CMV viremia.

to verify the nature of such 200-nm-sized particles, the following immunologica design was chosen. Two MAbs directed against 2 glycosylated polypeptides on the CMV envelope (Pereira et al. 1982) were indirectly bound to rabbit anti-mouse IgG, which was covaiently bound to the polystyrene microspheres in the first step. MAb-labelled microspheres were then incubated in serum or urine samples from patients with suspected CMV infection or in supernatants from tissue cultures. The beads were retained on the surface of a 0.6km filter after a l-h incubation and analyzed in the SEM. There was a distinct binding to such latex beads of about 200-nm-sized particles in 14 of 25 serum samples (Table 1 and Fig. 1) and in 23 of 2.5 urine samples (Table 1 and Fig. 2) obtained from bone marrow- or kidneytransplanted patients. Buffy coat and urine from these patients were seeded on embryonic fibroblasts, and CMV was isolated from the blood in 15 patients and in the urine in 25 patients (Table 1). Consecutive tests for a CMV-specific antibody response indicated ongoing CMV infection in 20 of these patients. No binding of particles to the beads was noticed when microspheres (3.92 pm) lacking the MAbs directed against CMV were incubated in serum samples con-

60

TABLE 2 Specific binding of virus, produced in tissue cultures, to beads labelled with anti-CMV antibodies’. No of isolates

Type of virus2

Tissue culture cells

Binding to MAb-labelled beads3

I

6 3 2 1 2 2 1 5 1 1 7

HSV not typed HSV type 1 HSV type 2 HSV type 2 Adenovirus Influenza A virus Influenza B virus Parainfluenza type 3 Varicellae virus Variceilae virus Varicelfae virus CMV

2 2

HIV EBV

GMK cells GMK cells GMK cells Vero cells Hela cells Mdck cells Mdck cells Mdck cells Fibroblasts Human embryonic libroblasts Mrc 5 fibroblasts 5 human embryonic fibroblasts 2 Mrc 5 ceils H9 human T-lymphocyte cells B 95-8 cells

O/l 016 013 I/2 binding of 100-nm particles Oil 012 012 O/l O/5 l/l binding of 170-nm particles 011 4/S 212 012 012

‘Latex beads were indirectly labelled with MAbs specific for human CMV (see Methods). ‘One hundred ~1 of centrifuged tissue culture supernatant was incubated with 1 x 10“ MAb-labelled 3.92pm latex beads. ‘At least 1 x 103beads were examined in each experiment in the SEM. All samples were analyzed blindly.

taining CMV particles (Fig. 3). In addition, no virus particles bound to the MAblabelled beads in 15 serum samples obtained from nonsymptomatic transplanted CMV-seropositive patients. Buffy coat samples from these 15 patients were also negative for CMV isolation (Table l), However, 10 of these patients excreted CMV in their urine, as determined by tissue culture and by filtration (Fig. 2, Table 1). No particles of the size of CMV could be found on the MAb-labelled beads or on the filter surface in serum samples from 25 CMV-seronegative healthy individuals, nor in urine samples from 20 CMV-seronegative individuals either. These samples were both filtered directly onto 0.2~pm filters and incubated with anti-CMV MAblabelled beads which were collected on 0.6~km polycarbonate filters (Fig. 4). Thus, the presence of CMV particles in serum reflected a viremia rather than a silent persistent virus replication state (Table 1). For control of specificity, the anti-CMV MAb-labelled microspheres were incubated with different viruses isolated from tissue cultures representing 36 virus strains of 9 virus types, not including CMV (Table 2). In addition 20 original patient samples were examined from either urine, serum, and saliva, or vesicle contents from which successful virus cultures (EBV, HSV type 1 and 2, and varicella) were performed (Table 1). No false binding to the beads of 1.5~ZOO-nm virus particles was observed in the SEM after filtration apart from 1 of 7 varicella-zoster isolates (Table 1 and 2). These samples were also examined blindly.

Fig. 2. Ten thousand anti-CMV-coated beads (3.92 wrn) were incubated in 200 ~1 of centrifuged urine (1.500 x g, 10 min) for 1 h at 37°C before they were accumulated on a 0.6 pm filter. The urine was positive for CMV by tissue culture.

Discussion

The results of this study indicate that filtration in combination with immune SEM is not only a rapid 2-h assay but also has a high specificity. Virus particles were identified in supernatants from 6 out of 7 tissue cultures for CMV grown in embryonic fibroblasts. The nonreacting sample mainly contained approx. lOO+,m-sized particles, probably nucleocapsides. These were not bound to the beads but were found on the polycarbonate filter surface. Only 1 varicella strain out of 36 various virus samples did cross-react with the indirect anti-CMV MAb-labelled beads. The intermediate rabbit anti-mouse IgG antisera was absorbed against human Ig in order to decrease the risk of false-positive binding of other virus types, covered by endogenously produced IgG, to the microspheres. No false binding was registered in 65 clinical samples, control serum, vesicle contents, or body fluids containing the other herpes virus types. Thus, the 2 MAbs, CH 12 p and CH 1620, must be highly specific for CMV. They are directed against the glycosylated envelope polypeptides E and F, respectively. However, these antigenic determinants must be type common and expressed frequently in various CMV strains since 92.5% of the clinical samples did bind to one or both of these

Fig. 3. Microspheres (3.92 km) treated in the same way, but without addition of the 2 anti-CMV MAbs, were incubated in 100 )LI serum from a patient with a positive virus isolation from buffy coat. No binding of CMV particles to the beads was detected when they were retained on the filter.

epitopes, as compared to CMV isolation by tissue culture. This is particularly promising in view of the fact that Chou (1986) has recently shown by restriction enzyme analysis of viral DNA that a great variation exists between various clinical CMV isolates. The clinical material presented was obtained from transplanted patients being immunosuppressed by Cyclosporin A and steroid treatment. Ten of 1.5 viremic patients had a primary CMV infection and 5 had serological evidence of a reactivated infection. The immune SEM filtration method detected CMV particles in the same frequency in both these groups. CMV was also identified by the filtration method in the urine in 8 out of 10 patients with asymptomatic CMV excretion. Risk for false-negative results can appear if endogenously produced specific antibodies block all the antigenic determinants on the extracellular CMV particles and thereby reduce the possibility of binding to the Mab-labelled beads. However, the sensitivity of the filtration method in the patient groups studied was comparable to virus isolation by tissue culture. It remains to be studied whether the filtration method also can be used to identify CMV infections in immunocompetent individuals. These patients may have lower virus titers and a more pronounced specific

Fig. 4. MAb-CMV-labelled 3.92.km microspheres incubated seronegative individual, and accumulated on a 0.6pm filter. beads or on the filter.

in 100 ~1 serum from a normal CMVNo particles were detected around the

humoral and cellular immune response, reducing the possibilities of identifying extracellular antigenic CMV particles. In these experiments, sample volumes of 100-200 ~1 were used, but the sensitivity of the immune SEM filtration method can perhaps be increased by amplifying the sample volume. No differences were demonstrated when comparing l-h incubation of the beads in 37°C with 12 h in + 4”C, (data not shown). The immune SEM method may also be used in the future for quantification of extracellular virus in various body fluids, simply by counting the number of virus particles bound to a certain number of antibody-labelled beads. The method described here may be used in general to detect virus in ail body fluids. Among the various methods for virus isolation by tissue culture, it has the advantage of not only being more rapid, but also of not being negatively affected by antiviral substances that may inhibit culture conditions and cause a false-negative isolation. This combination of binding virus to MAb-labelled beads for specificity, filtering for enrichment, SEM for size, as well as quantity and rapid analysis bings viral diagnosis to a new stage of development.

64

Acknowledgements

This work was supported by grants from the Swedish Medical Research Council, the Swedish Medical Association and the Swedish Board for Technical Development. We thank Dr. Leonore Pereira, University of California, San Francisco, for kindly providing MAbs against human CMV, the staff at the Transplantation Department, Huddinge Hospital, for providing all clinical samples and the staff at the National Bacteriological Laboratory, Stockholm, for performing all the virus isolations and serology tests.

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