JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1975, p. 332-336 Copyright ©) 1975 American Society for Microbiology
Vol. 2, No. 4 Printed in U.S.A.
Demonstration of Immunoglobulin G Receptors Induced by Human Cytomegalovirus TORU FURUKAWA,* ELIZABETH HORNBERGER, SADATOSHI SAKUMA, AND STANLEY A. PLOTKIN The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
Received for publication 11 July 1975
Human cytomegalovirus induced a new immunoglobulin G receptor in human fibroblasts. The immunoglobulin G receptor was well localized in the perinuclear region at 48 h postinfection, and antiviral agents blocked its synthesis. The immunoglobulin G receptor bound immunoglobulin G of man and several other species. It may be a source of error in the performance of indirect fluorescence tests for human cytomegalovirus antibody. Human cytomegalovirus (HCMV) infection induces various morphological and biochemical changes in cells (2, 7). The most distinct morphological change in infected human fibroblasts is the formation of nuclear and cytoplasmic inclusion bodies. The unique nuclear inclusion bodies (NI) have been the most reliable marker of HCMV infection and have been extensively studied by electron microscopy (3, 4). However, little is known concerning the nature of the cytoplasmic inclusion bodies (CI). Many serological methods, including complement fixation (CF), indirect hemagglutination, precipitin, and neutralization tests have become available to measure antibody for HCMV. The indirect immunofluorescent staining technique has been one of the fastest and most sensitive tests for measuring antibody levels and for identifying HCMV. During time course experiments, in which cells were infected with HCMV and stained by immunofluoresence, it was noted that HCMV induced an immunoglobulin G (IgG) receptor on the infected cell. In this paper we describe the characteristics of the IgG receptor and its potential clinical significance. While this work was in progress Keller et al. described a similar phenomenon in a published abstract (R. Keller, R. Pettchel, J. Goldman, and M. Goldman, Abstr. Annu. Meet. Am. Soc. Microbiol., 1975, S264, p. 257). MATERIALS AND METHODS Virus. The Towne strain of HCMV was used in these experiments. The history of this strain has been described elsewhere (1). Cells. WI-38 human diploid cells obtained from L. Hayflick were used between the 20th and 30th passage levels. The cells were grown in Eagle minimal essential medium (MEM) supplemented for growth or maintenance with 10% or 2% fetal calf serum, respectively. 332
Infectivity assay. The plaque assay, according to the method of Wentworth and French (10), was used. Inoculated cultures were incubated in a CO2 incubator under a final concentration of 0.3% agarose. The plaques were counted by microscopic observation 14 days postinfection (PI), after staining with methylene blue. Cytological study. To observe cytopathology, cells were grown on coverslips in petri dishes. After the cells were fixed with Bouin fixative, they were stained with 5% May-Greenwald-Giemsa or hematoxylin eosin. CF test. The CF test was performed using the microtiter technique with 2 U of antigen obtained from Flow Laboratories (Rockville, Md.) and 2 U of complement. Sera. Sera were collected for clinical diagnosis from various sources and stored at -20 C until use. All sera were heat inactivated for 30 min at 57 C. The sera used were from children, newborn to 5 years of age. Cord sera were obtained from the Department of Obstetrics, University of Pennsylvania. Immunofluorescent study. For the immunofluorescent study, cells grown on Lab Tek chamber slides or cover slips were fixed with cold acetone for 10 min and air dried. The following fluorescein isothiocyanate (FITC) conjugates were used: (i) rabbit anti-human gamma globulin prepared by Behring Diagnostics (Laurel Springs, N.J.); (ii) rabbit antihamster gamma globulin prepared by Cappel Laboratories (Downingtown, Pa.); (iii) rabbit anti-guinea pig gamma globulin prepared by Cappel Laboratories; (iv) goat anti-rabbit gamma globulin prepared by Cappel Laboratories; (v) rabbit anti-human IgM globulin prepared by Behring Diagnostics; and (vi) human IgG and human IgM obtained from Cappel Laboratories. Method of fluorescent antigen staining. Either freshly fixed cells or those stored for up to 3 weeks at -20 C were used for fluorescent antibody. Preparations were dipped in phosphate-buffered saline (PBS), drained, and flooded with primary antiserum (HCMV, CF positive; HCMV, CF negative) or PBS. These were incubated at 37 C for 30 min, washed three times with PBS, and flooded with a secondary
VOL. 2, 1975
IgG RECEPTORS IN HUMAN CYTOMEGALOVIRUS
antiserum which was FITC conjugated. After a second 30-min incubation, the preparations were again washed three times with PBS and flooded with 0.1% Evan blue in deionized water. After 7 min of incubation at room temperature, slides were washed twice with deionized water, air dried, and mounted in Elvanol. The preparations were examined with a Leitz microscope equipped with a HBC 200 mercury lamp and a dry dark field condensor. A primary BG3 filter and a secondary 44 filter were used. Hemadsorption (HA). The method used was essentially that described by Watkins (9) and Furukawa et al. (2a). Briefly, sheep erythrocytes were sensitized by adding 0.01 ml of rabbit anti-sheep erythrocyte serum to 1.0 ml of a 4% suspension of sheep erythrocytes in MEM. After incubation at 37 C for 1 h, the erythrocytes were washed three times in PBS and resuspended in MEM to a final concentration of 1%. At various intervals PI, cover slips were washed, and 1% sensitized sheep erythrocytes were added. After incubation for 2 h at 37 C, the cover slips were washed three times with PBS, stained with Giemsa, and examined microscopically. Chemicals. Cytosine arabinoside and cycloheximide were purchased from Nutritional Biochemicals Corp. (Cleveland, Ohio).
RESULTS WI-38 cells were infected at a multiplicity of 10 plaque-forming units (PFU) of virus per cell. At various intervals the cultures were assayed for HA, cell-associated infectious virus, and the presence of inclusion bodies. The results of these tests are shown in Fig. 1. Viral replication was evident at about 48 h PI, and cellassociated virus reached a peak 5 days PI. NI were not demonstrated by ordinary staining until 3 days PI. Immunofluorescent staining with CF-positive sera revealed that faint nu-
HOURS POST INFECTION
FIG. 1. Time course of appearances of HA and immunofluorescent antigens in relation with growth cycle of HCMV. Symbols: 0, infectivity; A, HA; A, NI immunofluorescence; OI, CI immunofluorescence.
333
clear staining appeared 9 to 24 h PI. At 48 h PI immunofluorescent antigens were seen in the nucleus in a honeycomb-like pattern. Fluorescence was also seen in the perinuclear region. At 72 h PI, antigen was present in pronounced NI and, additionally, in diffusely stained but well-localized perinuclear CI (Fig. 2A). These inclusions were also demonstrated by hematoxylin eosin staining. HA was observed in 60% of the cells at 48 h PI and reached a peak of 78% at 72 h PI. In contrast to these findings with CF-positive sera, only cytoplasmic immunofluorescent staining, coinciding with CI, was seen with CFnegative sera (Fig. 2B). Effect of antiviral agents on cytoplasmic inclusions. Confluent monolayers of WI-38 cells were infected with HCMV at a multiplicity of 1 PFU per cell. After 1 h of adsorption the medium was replaced with MEM containing 2% fetal calf serum and cycloheximide (2 ,g/ml) or cytosine arabinoside (100 A.tg/ml). At 3 days PI, cells were fixed with acetone and stained using the indirect immunofluorescence technique. In cells treated with either inhibitor the NI were not stained; in addition, CI were not demonstrated with sera either positive or negative for HCMV antibody. In cytosine arabinoside-treated cells, faint diffuse nuclear staining was seen corresponding to the early antigen reported by The et al. (8). Under these conditions (treatment with cytosine arabinoside or cycloheximide) more than 90% of infectious virus production was inhibited (5, 8). These results indicated that CI were induced by HCMV infection and could be inhibited by inhibitors of either DNA or protein synthesis. Antibody distribution among human sera. Selected sera from infants and children, newborn to 5 years of age, and umbilical cord sera were assayed for immunofluorescent staining of CI or NI and by CF tests. All CF-positive sera stained for CI and NI, and the titers varied from 1:80 to 1:320 for NI and 1:220 to 1:5028 for CI. In contrast to these findings, CF-negative sera showed only staining of CI. The titers were comparable to those of CF-positive sera (Fig. 3). The immunofluorescent antibody titer for NI was negative when the CF titer was negative. CF-positive sera also had higher staining titers against CI than against NI, but no difference in staining titers for CI was observed between CF-positive and CF-negative sera. These data indicated that "antibody" for CI might be some type of normally existing Ig instead of specific antibody for HCMV. Therefore, the subsequent experiments were carried out to determine the specificity of CI staining.
334
FURUKAWA ET AL.
J. CLIN. MICROBIOL.
FIG. 2. Fluorescent micrograph of infected WI-38 human diploid cells by HCMV (X400). (A) Indirect staining by CF-positive human serum; (B) indirect staining by CF-negative human serum.
Infected monolayers on cover slips were fixed with cold acetone at 3 days PI. Immunofluorescent staining was performed with standard CFnegative serum by the indirect method using labeled anti-human IgG and IgM. Direct staining was also performed with labeled human IgG and IgM using PBS instead of intermediate serum. The results are summarized in Table 1. Only anti-IgG fluorescent sera stained CI by
both the direct and indirect methods. These results suggested that the binding site(s) of CI was an IgG receptor for human sera. The same type of experiments were carried out using hamster, rabbit and guinea pig sera. All were positive, indicating that CI had a common receptor for the IgG of different species. The presence of an IgG receptor was also confirmed by a blocking test (Table 2). Fluores-
VOL. 2, 1975
335
IgG RECEPTORS IN HUMAN CYTOMEGALOVIRUS '320
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00000
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0
160
160
-
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Vol. 2, No. 4 Printed in U.S.A.
Demonstration of Immunoglobulin G Receptors Induced by Human Cytomegalovirus TORU FURUKAWA,* ELIZABETH HORNBERGER, SADATOSHI SAKUMA, AND STANLEY A. PLOTKIN The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
Received for publication 11 July 1975
Human cytomegalovirus induced a new immunoglobulin G receptor in human fibroblasts. The immunoglobulin G receptor was well localized in the perinuclear region at 48 h postinfection, and antiviral agents blocked its synthesis. The immunoglobulin G receptor bound immunoglobulin G of man and several other species. It may be a source of error in the performance of indirect fluorescence tests for human cytomegalovirus antibody. Human cytomegalovirus (HCMV) infection induces various morphological and biochemical changes in cells (2, 7). The most distinct morphological change in infected human fibroblasts is the formation of nuclear and cytoplasmic inclusion bodies. The unique nuclear inclusion bodies (NI) have been the most reliable marker of HCMV infection and have been extensively studied by electron microscopy (3, 4). However, little is known concerning the nature of the cytoplasmic inclusion bodies (CI). Many serological methods, including complement fixation (CF), indirect hemagglutination, precipitin, and neutralization tests have become available to measure antibody for HCMV. The indirect immunofluorescent staining technique has been one of the fastest and most sensitive tests for measuring antibody levels and for identifying HCMV. During time course experiments, in which cells were infected with HCMV and stained by immunofluoresence, it was noted that HCMV induced an immunoglobulin G (IgG) receptor on the infected cell. In this paper we describe the characteristics of the IgG receptor and its potential clinical significance. While this work was in progress Keller et al. described a similar phenomenon in a published abstract (R. Keller, R. Pettchel, J. Goldman, and M. Goldman, Abstr. Annu. Meet. Am. Soc. Microbiol., 1975, S264, p. 257). MATERIALS AND METHODS Virus. The Towne strain of HCMV was used in these experiments. The history of this strain has been described elsewhere (1). Cells. WI-38 human diploid cells obtained from L. Hayflick were used between the 20th and 30th passage levels. The cells were grown in Eagle minimal essential medium (MEM) supplemented for growth or maintenance with 10% or 2% fetal calf serum, respectively. 332
Infectivity assay. The plaque assay, according to the method of Wentworth and French (10), was used. Inoculated cultures were incubated in a CO2 incubator under a final concentration of 0.3% agarose. The plaques were counted by microscopic observation 14 days postinfection (PI), after staining with methylene blue. Cytological study. To observe cytopathology, cells were grown on coverslips in petri dishes. After the cells were fixed with Bouin fixative, they were stained with 5% May-Greenwald-Giemsa or hematoxylin eosin. CF test. The CF test was performed using the microtiter technique with 2 U of antigen obtained from Flow Laboratories (Rockville, Md.) and 2 U of complement. Sera. Sera were collected for clinical diagnosis from various sources and stored at -20 C until use. All sera were heat inactivated for 30 min at 57 C. The sera used were from children, newborn to 5 years of age. Cord sera were obtained from the Department of Obstetrics, University of Pennsylvania. Immunofluorescent study. For the immunofluorescent study, cells grown on Lab Tek chamber slides or cover slips were fixed with cold acetone for 10 min and air dried. The following fluorescein isothiocyanate (FITC) conjugates were used: (i) rabbit anti-human gamma globulin prepared by Behring Diagnostics (Laurel Springs, N.J.); (ii) rabbit antihamster gamma globulin prepared by Cappel Laboratories (Downingtown, Pa.); (iii) rabbit anti-guinea pig gamma globulin prepared by Cappel Laboratories; (iv) goat anti-rabbit gamma globulin prepared by Cappel Laboratories; (v) rabbit anti-human IgM globulin prepared by Behring Diagnostics; and (vi) human IgG and human IgM obtained from Cappel Laboratories. Method of fluorescent antigen staining. Either freshly fixed cells or those stored for up to 3 weeks at -20 C were used for fluorescent antibody. Preparations were dipped in phosphate-buffered saline (PBS), drained, and flooded with primary antiserum (HCMV, CF positive; HCMV, CF negative) or PBS. These were incubated at 37 C for 30 min, washed three times with PBS, and flooded with a secondary
VOL. 2, 1975
IgG RECEPTORS IN HUMAN CYTOMEGALOVIRUS
antiserum which was FITC conjugated. After a second 30-min incubation, the preparations were again washed three times with PBS and flooded with 0.1% Evan blue in deionized water. After 7 min of incubation at room temperature, slides were washed twice with deionized water, air dried, and mounted in Elvanol. The preparations were examined with a Leitz microscope equipped with a HBC 200 mercury lamp and a dry dark field condensor. A primary BG3 filter and a secondary 44 filter were used. Hemadsorption (HA). The method used was essentially that described by Watkins (9) and Furukawa et al. (2a). Briefly, sheep erythrocytes were sensitized by adding 0.01 ml of rabbit anti-sheep erythrocyte serum to 1.0 ml of a 4% suspension of sheep erythrocytes in MEM. After incubation at 37 C for 1 h, the erythrocytes were washed three times in PBS and resuspended in MEM to a final concentration of 1%. At various intervals PI, cover slips were washed, and 1% sensitized sheep erythrocytes were added. After incubation for 2 h at 37 C, the cover slips were washed three times with PBS, stained with Giemsa, and examined microscopically. Chemicals. Cytosine arabinoside and cycloheximide were purchased from Nutritional Biochemicals Corp. (Cleveland, Ohio).
RESULTS WI-38 cells were infected at a multiplicity of 10 plaque-forming units (PFU) of virus per cell. At various intervals the cultures were assayed for HA, cell-associated infectious virus, and the presence of inclusion bodies. The results of these tests are shown in Fig. 1. Viral replication was evident at about 48 h PI, and cellassociated virus reached a peak 5 days PI. NI were not demonstrated by ordinary staining until 3 days PI. Immunofluorescent staining with CF-positive sera revealed that faint nu-
HOURS POST INFECTION
FIG. 1. Time course of appearances of HA and immunofluorescent antigens in relation with growth cycle of HCMV. Symbols: 0, infectivity; A, HA; A, NI immunofluorescence; OI, CI immunofluorescence.
333
clear staining appeared 9 to 24 h PI. At 48 h PI immunofluorescent antigens were seen in the nucleus in a honeycomb-like pattern. Fluorescence was also seen in the perinuclear region. At 72 h PI, antigen was present in pronounced NI and, additionally, in diffusely stained but well-localized perinuclear CI (Fig. 2A). These inclusions were also demonstrated by hematoxylin eosin staining. HA was observed in 60% of the cells at 48 h PI and reached a peak of 78% at 72 h PI. In contrast to these findings with CF-positive sera, only cytoplasmic immunofluorescent staining, coinciding with CI, was seen with CFnegative sera (Fig. 2B). Effect of antiviral agents on cytoplasmic inclusions. Confluent monolayers of WI-38 cells were infected with HCMV at a multiplicity of 1 PFU per cell. After 1 h of adsorption the medium was replaced with MEM containing 2% fetal calf serum and cycloheximide (2 ,g/ml) or cytosine arabinoside (100 A.tg/ml). At 3 days PI, cells were fixed with acetone and stained using the indirect immunofluorescence technique. In cells treated with either inhibitor the NI were not stained; in addition, CI were not demonstrated with sera either positive or negative for HCMV antibody. In cytosine arabinoside-treated cells, faint diffuse nuclear staining was seen corresponding to the early antigen reported by The et al. (8). Under these conditions (treatment with cytosine arabinoside or cycloheximide) more than 90% of infectious virus production was inhibited (5, 8). These results indicated that CI were induced by HCMV infection and could be inhibited by inhibitors of either DNA or protein synthesis. Antibody distribution among human sera. Selected sera from infants and children, newborn to 5 years of age, and umbilical cord sera were assayed for immunofluorescent staining of CI or NI and by CF tests. All CF-positive sera stained for CI and NI, and the titers varied from 1:80 to 1:320 for NI and 1:220 to 1:5028 for CI. In contrast to these findings, CF-negative sera showed only staining of CI. The titers were comparable to those of CF-positive sera (Fig. 3). The immunofluorescent antibody titer for NI was negative when the CF titer was negative. CF-positive sera also had higher staining titers against CI than against NI, but no difference in staining titers for CI was observed between CF-positive and CF-negative sera. These data indicated that "antibody" for CI might be some type of normally existing Ig instead of specific antibody for HCMV. Therefore, the subsequent experiments were carried out to determine the specificity of CI staining.
334
FURUKAWA ET AL.
J. CLIN. MICROBIOL.
FIG. 2. Fluorescent micrograph of infected WI-38 human diploid cells by HCMV (X400). (A) Indirect staining by CF-positive human serum; (B) indirect staining by CF-negative human serum.
Infected monolayers on cover slips were fixed with cold acetone at 3 days PI. Immunofluorescent staining was performed with standard CFnegative serum by the indirect method using labeled anti-human IgG and IgM. Direct staining was also performed with labeled human IgG and IgM using PBS instead of intermediate serum. The results are summarized in Table 1. Only anti-IgG fluorescent sera stained CI by
both the direct and indirect methods. These results suggested that the binding site(s) of CI was an IgG receptor for human sera. The same type of experiments were carried out using hamster, rabbit and guinea pig sera. All were positive, indicating that CI had a common receptor for the IgG of different species. The presence of an IgG receptor was also confirmed by a blocking test (Table 2). Fluores-
VOL. 2, 1975
335
IgG RECEPTORS IN HUMAN CYTOMEGALOVIRUS '320
0 0 000
LL
0
ea * 0000
00000
> 320
000 @0.
U.
0
0
160
160
-
z
Z (I)
J
