Identification of Herpes Simplex and Vaccinia Viruses in Corneal Cell Cultures with Immunoperoxidase: a Light and Electron Microscopic Study*
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Identification of herpes simplex and vaccinia viruses in corneal cell cultures with immunoperoxidase: a light and electron microscopic study* Alan L. Shabo, John C. Vetricciani, and Ruth L. Kirschstein Peroxidase-labeled antibody (pooled human immune globulin) was employed to localize herpes simplex and vaccinia viruses in corneal cells maintained in culture. With the light microscope, intranuclear and paranuclear staining were noted in cells infected with herpes simplex while intracytoplasmic staining was found in cells infected with vaccinia. Electron microscopy confirmed the differential, specific viral antigen labeling by peroxidase marker. The immunoperoxidase method, utilizing more specific antibodies in conjunction with elec- tron microscopy, could presumably localize viral precursor antigens during different phases of infection. The relative advantages of this method in comparison to immunofluorescence and immunoferritin are discussed. Due to the specific staining achieved and its dual micro- scopic potential, we believe that the immunoperoxidase method may be useful as a diagnos- tic tool and could assist in understanding the immunopathology of corneal viral infections. Key words: immunoperoxidase, substrate, reaction product, virion, herpes simplex, vaccinia. JLhe immunopathology of cells infected cence has proved to be a valuable re- with herpes simplex or vaccinia viruses search and diagnostic tool.1'13 With the has been examined by various methods de- electron microscope, the immunoferritin signed to localize intracellular viral anti- technique has been employed widely, par- gens. For light microscopy, immunofluores- ticularly in studies of herpes simplex virus.9"11 However, until recently12"14 no single method was available for both light From the Laboratory of Pathology, Division of and electron microscopic localization of Biologies Standards, National Institutes of intracellular antigens. Horseradish peroxi- Health, Bethesda, Md. 20014. dase is an enzyme which, following expo- Manuscript submitted for publication May 3, 1973; sure to the appropriate substrate, forms an manuscript accepted for publication June 21, electron-dense reaction product. The en- 1973. zymatic activity of peroxidase is retained Reprint requests: Alan L. Shabo, M.D. Jules Stein after conjugation to antibody and the na- Eye Institute, 800 Westwood Plaza, Los Angeles, Calif. 90024. ture of the substrate reaction allows for "Presented in part at the 1972 ARVO Meeting the specific localization of reaction product Sarasota, Fla. with the light and electron microscopes. 839 Downloaded from iovs.arvojournals.org on 09/24/2021 840 Shabo, Petricciani, and Kirschstein Investigative Ophthalmology November 1973 This report describes the application of Application of peroxidase-labeled antibodies. the immunoperoxidase method to the iden- Light microscopy. Infected cells with 2 to 3 + CPE were fixed in situ for 15 minutes in cold ace- tification of herpes simplex and vaccinia tone and then rinsed without air drying in three viruses in corneal cell cultures. Data are changes of PBS. The peroxidase-antibody con- presented which suggest that this method jugate (1 ml. of a 1:2 dilution) was applied to could benefit immunopathologic studies of the cells for 30 minutes in a moist chamber at corneal viral infections. 37° C. After exposure to this conjugate, the cells were rinsed again in three changes of PBS and incubated for 15 minutes in 25 ml. of substrate Materials and methods solution at room temperature. The substrate con- sisted of 3-3' diaminobenzidine tetrahydrochloride Viruses. Herpes simplex virus (HSV), strain (0.5 mg. per milliliter) in 0.05 M. Tris-HCl buffer HF-490, was obtained from Dr. G. Silver, Micro- at pH 7.6 containing 0.01 per cent hydrogen biological Associates, and a pool was prepared peroxide.10 Following the substrate incubation, the from this material in rabbit kidney cells. Vaccinia cells were rinsed in three more changes of PBS, virus, strain CV1-78,15 was obtained from Dr. stained with Giemsa, and examined with the light R. Dunlap, Division of Biologies Standards microscope. (DBS), National Instiutes of Health. Infection of cell cultures. An epithelial-like cell Electron microscopy. Infected cells with 1 to 2 line derived from rabbit cornea, SIRC,16 was ob- + CPE were fixed in situ for 1 hour in cold 4 tained from Mrs. H. Hopps, Division of Biologies per cent paraformaldehyde containing 0.1 M. Standards, National Institutes of Health. Cultures cacodylate buffer and 0.01 per cent CaCl2 at pH were maintained in 250 ml. plastic Falcon flasks 7.3. The cells were rinsed three times in the same with medium MAB 87/31G supplemented with 5 buffer and allowed to wash overnight at 4° C. per cent fetal bovine serum without antibiotics at The next morning, the peroxidase-antibody con- 37° C. in a humidified incubator with 5 per cent jugate (20 ml. of a 1:2 dilution) was added for CCX' and air. Nearly confluent monolayers of SIRC 24 to 48 hours at 4° C. Following exposure to the cells were inoculated with HSV or vaccinia at a conjugate, the cells were rinsed in three changes multiplicity of infection (MOI) of 25 to 100. Fol- of 0.1 M. cacodylate buffer (pH 7.3) and fixed for lowing inoculation, the virus was allowed to ad- an additional hour in cold 4 per cent glutaralde- sorb for 1 hour at 37° C. and the cultures were hyde in the same buffer. Then the cells were then re-fed without rinsing with medium MAB rinsed three times with 0.1 M. cacodylate buffer 87/3 without serum. Cultures for electron micros- prior to a 15 minute incubation in substrate as copy were reacted with peroxidase-labeled anti- described for light microscopy. After the substrate body 24 to 48 hours after viral inoculation, while reaction, the cells were rinsed three additional cultures for light microscopy were studied at 24 times in buffer and postfixed for 1 hour in 1 per hours for vaccinia, and 18 to 72 hours for cent osmium tetroxide containing 0.1 M. cacody- HSV. late buffer at pH 7.3. The cells were scraped Preparation of peroxidase-antibody conjugate. gently from the flask surface, suspended in 75 Immune globulin (human, Lederle Laboratories) per cent ethyl alcohol, and centrifuged (1,800 x was used as the source of antibody to both vac- g for 5 minutes) into pellets. Finally, the pellets cinia and HSV. It had a protein content of 290 were rapidly dehydrated in ethyl alcohol and em- mg. per milliliter and neutralization titers of 1:1024 bedded in Araldite. Thin sections were cut, and 1:512, respectively, against HSV and vaccinia. stained with lead citrate and uranyl acetate, and Antibody was conjugated with horseradish peroxi- examined in the Hitachi HU11E electron micro- dase by a method modified after Nakane and scope. Pierce.12- 13 Details of this procedure have been Studies of control cell cultures. Uninfected cells described previously.18 In summary, horseradish and cells infected with HSV or vaccinia were peroxidase (Sigma Type II) was dissolved with an examined following application of the peroxidase- equal amount (by weight) of antibody protein antibody conjugate or substrate alone. An addi- in 0.5 M. cold Na2CO3) pH 10. This was followed tional control to demonstrate the specificity of the by the addition of 0.5 per cent p,p'-difluoro-m,m'- virus-antibody reaction consisted of exposing 20 dinitro-diphenyl sulfone (FNPS), stirring for 6 African green monkey cells, DBS-FCL-1, in- hours at 4° C, and overnight dialysis against fected with SV-40 virus to the peroxidase-labeled phosphate-buffered saline (PBS). The dialysate immune globulin plus substrate. was then centrifuged and the supernate combined with an equal volume of saturated ammonium Results sulfate. The precipitate was washed twice with 50 per cent saturated ammonium sulfate in PBS, Light microscopy. Fig. 1A illustrates then dissolved in PBS, and dialyzed against PBS SIRC cells that have been infected with overnight at 4° C. vaccinia virus and incubated in substrate Downloaded from iovs.arvojournals.org on 09/24/2021 Volume 12 Identification of herpes simplex and vaccinia viruses 841 Number 11 Fig. 1A. Corneal cells infected with vaccinia and incubated in substrate alone as a control. Multiple, small, intracytoplasmic inclusions or Guarnieri bodies (arrows) are present. Note the absence of large, dense intranuclear or intracytoplasmic inclusions. Giemsa, x700, oil immersion. Fig. IB. Following exposure to the peroxidase-antibody conjugate and substrate, cells infected with vaccinia reveal diffuse, dense-black intracytoplasmic inclusions of peroxidase reaction product that localize viral antigen. No intranuclear reaction product is present. Giemsa, x800, oil immersion. Fig, 1C. Cells infected with herpes simplex and incubated in the peroxidase-antibody con- jugate plus substrate demonstrate intranuclear and paranuclear staining by black peroxidase marker. Giemsa, x625, oil immersion. Downloaded from iovs.arvojournals.org on 09/24/2021 842 Shabo, Petricciani, and Kirschstein Investigative Ophthalmology November 1973 alone as a control. The corneal cells possess 2B illustrates the cytoplasm of a corneal round or oval nuclei and pale cytoplasm. cell which contains immature vaccinia In the cytoplasm of many cells, small, virions with incomplete membranes that round, eosinophilic inclusions (Guarnieri surround a focus of granularity (the "virus bodies)21 are identified that are character- factory"22). A mature virion (arrow) is also istic of vaccinial infections of epithelial shown and is composed of a central nu- cells. No large, dense intranuclear or in- cleoid enveloped by distinct membranes. tracytoplasmic inclusions are present, how- Fig. 3A demonstrates the nucleus and ever. The exposure to substrate does not adjacent cytoplasm of a cell infected with affect the appearance of these cells other herpes simplex that has been exposed to than to increase slightly the cellular den- the peroxidase-antibody conjugate and sub- sity. In Fig. IB, corneal cells have been strate. Several well-defined virions of her- infected with vaccinia virus and incubated pes simplex are present and labeled by in both the peroxidase-antibody conjugate dense, black, granular peroxidase reaction and substrate.