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Rapid immunofluorescent detection of a viral infection in tissue culture
Immunology Letters, 1 (1979) 11-13
© Elsevicr/North-Holland Biomedical Press
RAPID IMMUNOFLUORESCENT
D E T E C T I O N O F A V I R A L I N F E C T I O N IN
TISSUE CULTURE H. H. WEETALL, A. A. LUDERER, H. C. ORR* and P. G. PROBST* Corning Glass Works, Sullivan Science Park, Corning, N Y 14830 and *Food and Drug Administration, Division of Pathology, Bureau of Biologics, Bethesda, MD 20014, USA
(Accepted 15 .February 1979)
1. Summary Tissue culture cells infected with either DNA or RNA viruses showed specific immunofluorescence, 4 - 8 h after initiating infection, when stained with a FITC-conjugate of anti-purine immunoglobulins.
2. Introduction Use ofimmunofluorescence to demonstrate nuclear deoxyribonucleic acid (DNA) in cells is well documented [ 1 - 3 ] . However, to our knowledge there are no reports of the use of such antibodies to demonstrate viral infection in mammalian tissue culture cells. We have been able to detect virus-infected cells in vitro by fluorescent antibodies directed against purines. The value of this approach is the rapidity with which viral infections can be detected after initiation.
3. Materials and methods Antibodies to purine were elicited in rabbits by immunization with a purine-bovine serum albumin conjugate. The antisera produced in this manner had previously been shown to react with denatured DNA, but not with denatured ribonucleic acid (RNA) [4-6]. Fluorescein-isothiocyanate- (FITC) conjugated immunoglobulins were prepared from the ammonium sulfate precipitated antisera and purified on DEAE-cellulose or adsorbed with acetone liver powders 7.
For most studies, confluent monolayer cultures of a continuous line of vervent monkey liver cells in Lab-Tek Flaskette (Miles Laboratories, Inc.) were infected with a low multiplicity of either RNA or DNA viruses (table 1). In other experiments, bovine turbinate or primary rabbit kidney cells were used as substrates where appropriate. The cells were fixed at 4, 8, 16, 24 and 48 h post-infection, in cold acetone and stored at -20°C until used. Infected and noninfected controls were stained by the direct fluorescent antibody method employing an FITC-conjugate protein concentration of 0.5 mg/ml. The cells were counter-stained with 0.005% Evans blue dye in distilled water, mounted in buffered glycerol (pH 7.6) and immediately examined. For blocking experiments, cells were pretreated with either normal rabbit serum or unlabeled anti-purine serum at the proper dilutions. Cells were also pretreated with DNAse or RNAse (Worthington Biochemical Corp., Freehold, N J). For microscopy, a Leitz fluorescent microscope with an HBO Osram mercury lamp and dark-field condenser was used. The excitation filter was a BG-12 and the barrier filter was a Wratten No. 12.
4. Results Uninfected cells were negative regardless of length of incubation. Less than 1 cell per field showed any nuclear fluorescence (fig.l). All infected cells showed specific fluorescence beginning at 4 - 8 h post-infection. In most cases, the fluorescence was initially weak cytoplasmic fluorescence which increased in 11
Table 1 Viruses detected by anti-purine FITC-conjugate
RNA viruses Bovine viral diarrhea Measles Polio, Type I ECHO-11 DNA viruses Vaccina Herpes simplex virus Simian virus40
Strain (4)
Seed virus titer
Number of plaqueforming units per flaskette inoculated
NADL Edmonston (EL) Mahoney African monkey kidney derived
5.0 X 104 1.2 × 10 a 3.0 X l0 s
30 120 300
1.5 × l0 s
150
CVI-180 Rodanas Rhesus monkey kidney (gerber) derived
3.8 × 10 s 1.0 X 10 s
380 100
1.0 × l0 s
100
intensity with time o f infection. The RNA viruses in general showed stronger membrane and nuclear fluorescence than did the DNA viruses.
Figure 2 shows the differences observed in cells infected with herpes simplex virus, a DNA virus (fig.2A) and ECHO-11, an RNA virus (fig.2B) after 4 h incubation post-infection. The herpes simplex viral infection shows weak nuclear fluorescence rather granular in nature. The ECHO-11 infected cell shows bright cytoplasmic and nuclear fluorescence but unstained nucleoli. Treatment o f either virusinfected cells with unlabeled anti-purine serum or DNAse completely blocked the fluorescence;while treatment with RNAse had no effect.
5. Discussion
Fig.1. Uninfected control cells stained with FITC-conjugated anti-purine immunoglobulin. 12
The observed data suggests that by utilizing an anti-purine antiserum, one can detect early stages o f b o t h DNA- or RNA-type viral infections. It appears that the antiserum is reacting with DNA in infected cells associated with replicating virions. Since the R N A viruses utilized in these studies do not possess reverse transcriptase activity [ 8 - 1 1 ] , the fluorescence exhibited in these RNA virus-infected cells may reflect cellular DNA synthesis in response to viral infection. FITC conjugated anti-purine serum could have clinical application in rapid diagnosis o f viral infections as well as in studies o f laboratory virus-induced neoplasms.
Fig.2. A: Herpes simplex-infected cell stained with FITC-conjugated anti-purinc immunoglobulin 4 h after initial infection. B: ECttO-I 1 infected cell stained with FITC-conjugated anti-purine immunoglobulin 4 h after initial infection.
References [1 ] Coons, A. H. and Kaplan, M. G. (1950) J. Exp. Med. 91,1. [2] Klein, W. J., Beiser, S. M. and Erlanger, B. F. (1967) J. Exp. Med. 125, 61. [3] Rosenkranz, H. S., Erlanger, B. F., Tannenbaum, S. W. and Beiser, S. (1964) Science 145,282. [4] Weetall, H. H. and Weilky, N. (1965) Nature (London) 207,858. [5] Butler, V. P., Tannenbaum, S. W. and Beiser, S. M. (1965) J. Expt. Med. 121, 19. [ 6 ] Butler, V. P., Beiser, S. M., Erlanger, B. F., Tannenbaum, S. W., Cohen, S. and Bendich, A. (1962) DNAS 48, 1597.
[7] Campbell, D. H., Gurvey, J. S., Cramer, N. E. and Sussedorf, D. H. (1963) in: Methods in Immunology (W. A. Benjamin ed.) New York. [8] Albrecht, P. and Schumacher, H. P. (1972) Arch. Gesamte Virusforsch. 36, 23. [9] Kempe, H. C., Fulgeniti, V., Minamitani, V. and Shinefield, H. (1968) Pediatrics 42,980. [10] Pavon, P. R. and Ennus, F. A. (1977) J. Immunol. 118, 2167. [11] Fenner, F., McAuslan, B. R., Mims, C. A., Sambrook, J. and White, D. O. (1974) in: The Biology of Animal Viruses, p. 508, Academic Press, New York.
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© Elsevicr/North-Holland Biomedical Press
RAPID IMMUNOFLUORESCENT
D E T E C T I O N O F A V I R A L I N F E C T I O N IN
TISSUE CULTURE H. H. WEETALL, A. A. LUDERER, H. C. ORR* and P. G. PROBST* Corning Glass Works, Sullivan Science Park, Corning, N Y 14830 and *Food and Drug Administration, Division of Pathology, Bureau of Biologics, Bethesda, MD 20014, USA
(Accepted 15 .February 1979)
1. Summary Tissue culture cells infected with either DNA or RNA viruses showed specific immunofluorescence, 4 - 8 h after initiating infection, when stained with a FITC-conjugate of anti-purine immunoglobulins.
2. Introduction Use ofimmunofluorescence to demonstrate nuclear deoxyribonucleic acid (DNA) in cells is well documented [ 1 - 3 ] . However, to our knowledge there are no reports of the use of such antibodies to demonstrate viral infection in mammalian tissue culture cells. We have been able to detect virus-infected cells in vitro by fluorescent antibodies directed against purines. The value of this approach is the rapidity with which viral infections can be detected after initiation.
3. Materials and methods Antibodies to purine were elicited in rabbits by immunization with a purine-bovine serum albumin conjugate. The antisera produced in this manner had previously been shown to react with denatured DNA, but not with denatured ribonucleic acid (RNA) [4-6]. Fluorescein-isothiocyanate- (FITC) conjugated immunoglobulins were prepared from the ammonium sulfate precipitated antisera and purified on DEAE-cellulose or adsorbed with acetone liver powders 7.
For most studies, confluent monolayer cultures of a continuous line of vervent monkey liver cells in Lab-Tek Flaskette (Miles Laboratories, Inc.) were infected with a low multiplicity of either RNA or DNA viruses (table 1). In other experiments, bovine turbinate or primary rabbit kidney cells were used as substrates where appropriate. The cells were fixed at 4, 8, 16, 24 and 48 h post-infection, in cold acetone and stored at -20°C until used. Infected and noninfected controls were stained by the direct fluorescent antibody method employing an FITC-conjugate protein concentration of 0.5 mg/ml. The cells were counter-stained with 0.005% Evans blue dye in distilled water, mounted in buffered glycerol (pH 7.6) and immediately examined. For blocking experiments, cells were pretreated with either normal rabbit serum or unlabeled anti-purine serum at the proper dilutions. Cells were also pretreated with DNAse or RNAse (Worthington Biochemical Corp., Freehold, N J). For microscopy, a Leitz fluorescent microscope with an HBO Osram mercury lamp and dark-field condenser was used. The excitation filter was a BG-12 and the barrier filter was a Wratten No. 12.
4. Results Uninfected cells were negative regardless of length of incubation. Less than 1 cell per field showed any nuclear fluorescence (fig.l). All infected cells showed specific fluorescence beginning at 4 - 8 h post-infection. In most cases, the fluorescence was initially weak cytoplasmic fluorescence which increased in 11
Table 1 Viruses detected by anti-purine FITC-conjugate
RNA viruses Bovine viral diarrhea Measles Polio, Type I ECHO-11 DNA viruses Vaccina Herpes simplex virus Simian virus40
Strain (4)
Seed virus titer
Number of plaqueforming units per flaskette inoculated
NADL Edmonston (EL) Mahoney African monkey kidney derived
5.0 X 104 1.2 × 10 a 3.0 X l0 s
30 120 300
1.5 × l0 s
150
CVI-180 Rodanas Rhesus monkey kidney (gerber) derived
3.8 × 10 s 1.0 X 10 s
380 100
1.0 × l0 s
100
intensity with time o f infection. The RNA viruses in general showed stronger membrane and nuclear fluorescence than did the DNA viruses.
Figure 2 shows the differences observed in cells infected with herpes simplex virus, a DNA virus (fig.2A) and ECHO-11, an RNA virus (fig.2B) after 4 h incubation post-infection. The herpes simplex viral infection shows weak nuclear fluorescence rather granular in nature. The ECHO-11 infected cell shows bright cytoplasmic and nuclear fluorescence but unstained nucleoli. Treatment o f either virusinfected cells with unlabeled anti-purine serum or DNAse completely blocked the fluorescence;while treatment with RNAse had no effect.
5. Discussion
Fig.1. Uninfected control cells stained with FITC-conjugated anti-purine immunoglobulin. 12
The observed data suggests that by utilizing an anti-purine antiserum, one can detect early stages o f b o t h DNA- or RNA-type viral infections. It appears that the antiserum is reacting with DNA in infected cells associated with replicating virions. Since the R N A viruses utilized in these studies do not possess reverse transcriptase activity [ 8 - 1 1 ] , the fluorescence exhibited in these RNA virus-infected cells may reflect cellular DNA synthesis in response to viral infection. FITC conjugated anti-purine serum could have clinical application in rapid diagnosis o f viral infections as well as in studies o f laboratory virus-induced neoplasms.
Fig.2. A: Herpes simplex-infected cell stained with FITC-conjugated anti-purinc immunoglobulin 4 h after initial infection. B: ECttO-I 1 infected cell stained with FITC-conjugated anti-purine immunoglobulin 4 h after initial infection.
References [1 ] Coons, A. H. and Kaplan, M. G. (1950) J. Exp. Med. 91,1. [2] Klein, W. J., Beiser, S. M. and Erlanger, B. F. (1967) J. Exp. Med. 125, 61. [3] Rosenkranz, H. S., Erlanger, B. F., Tannenbaum, S. W. and Beiser, S. (1964) Science 145,282. [4] Weetall, H. H. and Weilky, N. (1965) Nature (London) 207,858. [5] Butler, V. P., Tannenbaum, S. W. and Beiser, S. M. (1965) J. Expt. Med. 121, 19. [ 6 ] Butler, V. P., Beiser, S. M., Erlanger, B. F., Tannenbaum, S. W., Cohen, S. and Bendich, A. (1962) DNAS 48, 1597.
[7] Campbell, D. H., Gurvey, J. S., Cramer, N. E. and Sussedorf, D. H. (1963) in: Methods in Immunology (W. A. Benjamin ed.) New York. [8] Albrecht, P. and Schumacher, H. P. (1972) Arch. Gesamte Virusforsch. 36, 23. [9] Kempe, H. C., Fulgeniti, V., Minamitani, V. and Shinefield, H. (1968) Pediatrics 42,980. [10] Pavon, P. R. and Ennus, F. A. (1977) J. Immunol. 118, 2167. [11] Fenner, F., McAuslan, B. R., Mims, C. A., Sambrook, J. and White, D. O. (1974) in: The Biology of Animal Viruses, p. 508, Academic Press, New York.
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