HSV-2 (4-6, 9). The resultant cell populations. contain HSV-specific cytoplasmic antigens (4, 5), HSV-specific surface antigens (6), and occasional

Transcription

1 JOURNAL OF VIROLOGY, Aug. 1973, p Copyright 1973 American Society for Microbiology Vol. 12, No. 2 Printed in U.S.A. Oncogenic Transformation of Hamster Embryo Cells After Exposure to Inactivated Herpes Simplex Virus Type 1 RONALD DUFF AND FRED RAPP Department of Microbiology, College of Medicine, The Milton S. Hershey Medical Center of The Pennsylvania State University, Hershey, Pennsylvania Received for publication 15 January 1973 The in vitro transformation of hamster embryo fibroblasts by herpes simplex virus type 1 (HSV-1) after exposure of the virus to UV irradiation is described. Cell transformation was induced by 2 out of 12 strains of HSV-1 that were tested for transforming potential. Cells transformed by the KOS strain of HSV-1 were not oncogenic when injected into newborn Syrian hamsters. However, cells transformed by HSV-1 strain induced tumors in 47% of the newborn hamsters injected. HSV-specific antigens were found in the cytoplasm of cells transformed by both virus strains. Sera from tumor-bearing hamsters contained HSV-1- and HSV-2-neutralizing antibodies as well as antibodies which reacted specifically with HSV antigens by the indirect immunofluorescence technique. Hamster oncornavirus antigens were not detected by immunofluorescence methods. These observations represent the first evidence of the oncogenic potential of HSV-1. Herpesviruses with oncogenic potential have been isolated from several animal species. For example, Marek's disease, a lymphoma of chickens, has been shown to be caused by a herpesvirus (2, 19). More recently, lymphomas and lymphocytic leukemia in monkeys have been induced by Herpesvirus saimiri (14, 16, 24). At least two herpesviruses of human origin have been implicated in cancer by epidemiological methods. The extensively studied Epstein- Barr virus has been associated with Burkitt's lymphoma of children as well as with the nononcogenic disease, infectious mononucleosis (7, 10-12). Herpes simplex virus type 2 (HSV-2) is the second human herpesvirus for which an association with human cancer has been suggested (18, 22). Women who have been exposed to HSV-2 via the genital route are more likely to develop carcinoma of the cervix than are women who have not been exposed to this virus. Herpes simplex virus type 1 (HSV-1) is also a possible candidate as an oncogenic virus due to its widespread occurrence throughout the human population and its genetic relationship to HSV-2 (1, 15). However, direct evidence suggesting that this virus has oncogenic potential has not been reported. transformation after infection by UV-irradiated HSV-2 (4-6, 9). The resultant cell populations contain HSV-specific cytoplasmic antigens (4, 5), HSV-specific surface antigens (6), and occasional incomplete HSV particles. HSV-specific neutralizing antibodies are also induced in the sera of tumor-bearing animals. This communication describes similar experiments with HSV-1 strains and describes some of the characteristics of the resulting transformed cells. MATERIALS AND METHODS Cells. Syrian HEF cells were extracted from 13- day-old embryos with trypsin. The embryos were obtained from the inbred LSH strain (obtained from Lakeview Hamster Colony, Newfield, N. J.). Embryos were treated with 0.25% trypsin for 15 min and then centrifuged to remove the trypsin after cell dispersion. Cells were suspended in Eagle medium supplemented with 10% fetal bovine serum, 10% tryptose phosphate broth, and 0.075% NaHCOs, and placed into 8-ounce glass precription bottles (approximately 240 ml) at a cell density of 5 x 106 cells per bottle. Rabbit kidney (RK) cells were obtained from weanling (21 to 28 day old) rabbit kidneys by a procedure nearly identical to that described for the preparation of the HEF cells. The RK cells were suspended in Eagle medium supplemented as described above and grown in plastic dishes (60 mm) for Recently, hamster embryo fibroblast (HEF) cells have been shown to undergo oncogenic use in routine HSV assays. 209

2 210 DUFF AND RAPP J. VIROL. Primary human embryo kidney (HEK) cells (obtained from HEM Research Inc., Rockville, Md.) were used after three cell culture passages in medium 199 supplemented with 10% fetal bovine serum, 10% tryptose phosphate broth, and 0.075% NaHCO3. The cells were grown in 8-ounce bottles (approximately 240 ml) when used for the preparation of stocks of HSV. Virus. HSV-1 strain KOS was isolated from a recurrent oral and was supplied by W. Rawls, Baylor College of Medicine, Houston, Texas. This strain of HSV-1 had been passed twice in HEK cells and once in RK cells and was passed three additional times in HEK cells before HEF transformation experiments. HSV-1 strain (obtained from C. A. Phillips, The University of Vermont College of Medicine, Burlington, Vt.) was isolated from a patient with chronic bronchitis and had been passed five times in WI-38 cells before being sent to our laboratory. This strain of HSV-1 was passed two additional times in HEK cells before use in transformation studies. HSV-2 strain 333 was obtained from W. Rawls after isolation from a primary genital. This strain of HSV-2, known to induce transformation of HEF (4, 5), was used as a prototype HSV-2 in these experiments. HSV-1 strain Patton was also obtained from W. Rawls after isolation from a recurrent oral. This strain of HSV-1 does not apparently transform HEF cells and was used as a control in these experiments. All HSV stocks were stored at -76 C in a Kelvinator freezer. Virus assay. The procedure for assaying HSV-1 and HSV-2 in RK cells has been previously described (20). Briefly, confluent monolayers of RK cells were inoculated with appropriate dilutions of herpesvirus. After virus absorption at room temperature for 1 h, the cell cultures were overlaid with Eagle medium containing 10% tryptose phosphate broth, 10% fetal bovine serum, 0.225% NaHCO3, and 1% methylcellulose. Four days after virus infection a 0.005% solution of neutral red was added to each petri dish, and the plaques were counted 4 h later. Immunofluorescence techniques. HSV-specific antigens were detected by indirect immunofluorescence methods. Monolayers of transformed cells grown on cover slips were washed three times in warm Tris buffer (ph 7.4) and air-dried. The cells were then fixed for 10 min in acetone, dried again, and exposed to HSV-specific antiserum for 30 min. The cells were then washed three times in Tris buffer. Anti-hamster gamma globulin prepared in a horse and previously conjugated to fluorescein isothiocyanate was placed on the cells. After 30 min of adsorption with fluorescein isothiocyanate-conjugated serum, the cells were washed three times in Tris buffer, air-dried, and mounted on glass slides. Specific fluorescence was detected by using a Zeiss microscope with a UV light source. HSV-1- and HSV-2-specific antibodies were prepared in weanling Syrian hamsters by using the method previously described (5). Neutralization of HSV. The plaque reduction technique was used for the detection of neutralizing antibodies directed against HSV-1 or HSV-2. A 0.5-ml amount of an appropriate serum dilution was added to 103 PFU of the virus to be tested. The serum was diluted 10-, 20-, 40-, and 80-fold. After incubation for 40 min at 37 C, 0.1 ml of the serum-virus mixture was placed on an RK cell monolayer and absorbed for 1 h. After absorption, the infected cultures were overlaid with Eagle medium containing 10% fetal bovine serum, 10% tryptose phosphate broth, 0.23% NaHCO3, and 1% methylcellulose. Four days later the cells were stained with neutral red and the plaques were counted. RESULTS Transformation of HEF cells by UV-irradiated HSV-1. The method used for transformation of HEF cells by UV-irradiated HSV-1 was very similar to the previously described method that resulted in the isolation of transformed cells after infection with UV-irradiated HSV-2 (4, 5). A cell-free suspension of HSV-1 (1.5 ml/sample) was exposed to UV irradiation (42 ergs per s per mm2) for 180 s, 240 s, 360 s, 480 s, and 600 s in a plastic petri dish (60 mm) with gentle agitation. No infective virus could be detected in HEF cells exposed to these preparations. Each sample of UV-irradiated HSV-1 was absorbed to HEF cells in suspension with shaking for 1 h at 37 C. Approximately 106 HEF cells were exposed to 106 HSV PFU which had been assayed in RK cells before UV irradiation. After virus absorption to the HEF cells in suspension, the exposed cells were placed in 250-ml plastic tissue culture bottles at a cell density of 3 x 105 cells per bottle and were grown for 14 days in medium 199 supplemented with 10% tryptose phosphate broth, 10% fetal calf serum, and 0.075% NaHCO3. The cell monolayers were then trypsinized, divided, and again grown in plastic tissue culture bottles. Two weeks later the cell monolayers were screened for transformed foci, and when transformed foci were observed they were isolated and grown into cell lines whenever possible. Twelve strains of HSV-1, representing separate human isolates, have been examined for transforming potential by this method (Table 1). Transformed cells were observed after exposure to two strains (KOS and ), and cell lines were developed from the transformed foci. The other 10 strains of HSV-1 did not induce morphological transformation. However, the possibility remains that these 10 HSV-1 strains might prove to have transforming potential that can be detected after more extensive testing. The morphology of the previously described HSV-2-transformed hamster embryo cells was predominantly a fibroblastic type with some cuboidal type cells and giant cells in the culture (4, 5). HEF cells transformed by the ( ) strain of HSV-1, as well as by the KOS strain (KOS-6-1) of HSV-1, induced cells

3 VOL. 12, 1973 TABLE 1. HSV-1 TRANSFORMATION OF HAMSTER CELLS Transforming potential of clinical isolates of HSV-1 Oncoge- Source ~~HEF nicity HSV-1 Source Site of cell of transstrain isola- isolation trans- fcoells ind activity" newborn hamsters Hill Houston Recurrent oral - - Haywood Houston Primary oral - - Seibert Houston Recurrent oral - - Edmonson Houston Recurrent oral - - Patton Houston Recurrent oral - - KOS Houston Recurrent oral Vermont Chronic bron- _ chitis Vermont Chronic bron- + + chitis Vermont Chronic bron- - - chitis Vermont Chronic bron- - - chitis Hickey Atlanta Vulva - - Lowe Atlanta Vulva - - avirus isolated in Houston was provided by W. Rawls, Baylor College of Medicine, Houston, Texas. Virus isolated in Vermont was provided by C. A. Phillips, University of Vermont, Burlington. Virus isolated in Atlanta was provided by A. Nahmias, Emory University College of Medicine, Atlanta, Georgia. b The method of transforming HEF cells by infection with UV-irradiated HSV-1 was described in the Materials and Methods. Each virus isolate was tested three times for transforming activity in HEF cells. A minus (-) signifies that no transformed foci appeared after exposure of HEF cells to UV-irradiated virus during the three tests. A plus (+) signifies that at least five transformed foci developed in HEF cells after virus infection in at least one test. with primarily an epithelial morphology (Fig. 1). In addition, cell lines transformed by HSV contained some cells with a fibroblastic morphology (Fig. 2). Both cell lines, KOS-6-1 and , contained numerous giant cells. However, neither cell line resembled the parental HEF cells from which they were derived. Neutralization characteristics of transforming HSV-1 strains. HSV-2 is the member of the herpes simplex virus group which has been most strongly implicated in cancer (18, 22). It was therefore necessary to test the HSV-1 strains which transformed HEF cells to determine whether these strains were antigenically HSV-1 (in addition to isolation from sites commonly associated with HSV-1). The plaque reduction method was used for this examination. The strains which were responsible for 211 transformation of HEF cells had the neutralizing characteristics which have been associated with type 1 viruses (Fig. 3). Both the KOS and the strains were neutralized by HSV-1 serum more efficiently than was HSV-2 strain 333. Furthermore, HSV-2 strain 333 was neutralized more efficiently by anti-hsv-2 serum than were the two HSV-1 isolates. The control, and thus far nontransforming, HSV-1 strain Patton was neutralized by anti-hsv-1 and anti-hsv-2 strain in a manner nearly identical to the KOS and strains. From these results it was concluded that HSV-1-KOS and HSV were antigenically type 1 viruses. Presence of HSV-specific antigens in cells transformed by HSV-1. HEF cells which had been transformed by HSV-1-KOS and HSV were examined for the presence of HSV-specific antigens in fixed cells by the indirect immunofluorescence technique. Transformed cells were treated with anti-hsv-1 serum, anti-hsv-2 serum, and normal hamster serum. Both KOS-6-1 and cells reacted with anti-hsv-1 and anti-hsv-2 serum with diffuse cytoplasmic staining in 1 to 10% of the cells (Fig. 4, Table 2). No specific fluorescence was detected when normal hamster serum was used. The specific immunofluorescence was observed in both the epithelial cell type and the fibroblastic cell type. Similar cytoplasmic antigens have been observed in HSV-2 transformed cells (4, 5). Oncogenicity of HSV-l-transformed cells in newborn hamsters. To determine whether the HSV-1-transformed cell lines were oncogenic, newborn Syrian hamsters were injected subcutaneously within 24 h after delivery with 2 x 105 transformed cells. Sixty hamsters were injected with the KOS-6-1 strain of cells, and none have developed tumors at the site of inoculation within 52 weeks after injection. However, 18 of 38 (47%) of the newborn hamsters injected with the cells developed tumors with latent periods ranging from 3 to 11 weeks. Preliminary pathological examination of the tumors revealed that they were not of the fibrosarcoma type which had been previously reported for the HSV-2-transformed hamster embryo cells (4, 5). Several tumors which were induced by the cell line were found to be pathologically similar to an adenocarcinoma. A complete pathological examination is in progress and will be published in a future report. HSV-specific antibodies in sera from tumor-bearing hamsters. Hamsters which developed a tumor as the result of injection of the HSV-2-transformed cell lines also developed neutralizing antibodies which were specific

4 )Lg+t Xr *tw 212 DUFF AND RAPP J. VIROL. AA b~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *-::- -e '}..... s.h 1fll #9*f FIG. 1. Photomicrograph of transformed hamster embryo fibroblast cells stained with hematoxylinii and eosin. The cells shown in this photomicrograph have a predominantly epithelial morphology. x300. W C.7 k.: I..w :1; T FIG. 2. Photomicrograph of transformed hamster embryo fibroblast cells stained with hematoxylin and eosin. Some cells shown in the photomicrograph have a predominantly fibroblastic morphology. x300.

5 VOL. 12, HSV-1 TRANSFORMATION OF HAMSTER CELLS C') 0 60 Cl) z n cc SERUM DILUTION FIG. 3. Neutralization of HSV-1 and HSV-2 by specific anti-hsv-i serum (0), specific anti-hsv-2 serum (-), and normal weanling hamster sera (0). The results are presented as the percentage of 1,000 infective HSV particles surviving incubation with an appropriate serum dilution for 40 min at 37 C. The specific antisera were prepared by the injection of UV-irradiated HSV-1 strain Patton or HSV-2 strain 333 into weanling Syrian hamsters. A, HSV-1 strain KOS; B, HSV-1 strain ; C, HSV-2 strain 333; D, HSV-1 strain Patton. against the HSV-2 (4, 5). Sera from hamsters bearing tumors induced by the injection of cells were also examined in a similar manner for the presence of HSV-neutralizing antibodies. Animals with a tumor induced by the injection of transformed cells developed relatively low levels of neutralizing antibodies directed against either HSV-1 or HSV-2 (Fig. 5). The low activity of these sera was probably a result of the short latent period of the cells in hamsters whose sera have been tested. Furthermore, the rapid growth of the tumor cells after injection into a newborn hamster may have induced a partial immune tolerance. The sera from tumor-bearing hamsters also reacted with HSV-1-transformed cells, HSV-2-transformed cells, and HSV-1- and HSV-2-infected HEF cells by the indirect immunofluorescence technique (Table 2). However, these sera did not react with normal HEF cells; sera from hamsters with tumors induced by HSV-2-transformed cells reacted with the HSV-1-transformed cells. Characteristics of cell lines developed from hamster tumors. Cells which were trypsinized and grown from tumors were examined to determine their morphology. Cells from the three tumors that were examined had two distinct morphological cells. The first tumor yielded cells consisting primarily of fibroblasts. Cells from the two other tumors were primarily epithelial in morphology. Cells of both morphological types that were isolated from hamster tumors contained antigens which reacted with either HSV-1- or HSV-2-specific antibodies, as was seen by using the indirect immunofluorescence technique (Table 3). However, no significant differences in the number of cells with specific HSV antigens could be determined for each morphological type. Absence of C-type particle antigens in HSV-l-transformed cells. The oncogene theory of neoplasia postulates that all oncogenic tumors are the result of the activation of an oncornavirus oncogene within the transformed cell (13). Previous examinations of HSV- 2-transformed cells have demonstrated that these cells do not contain detectable C-type

6 214 DUFF AND RAPP J. VIROL. FIG. 4. Immunofluorescence photomicrograph of HSV-J antigens localized in KOS-6-1-transformed hamster cells. x450. TABLE 2. Detection of HSV-specific antigens in HSV-transformed and infected HEF cells by immunofluorescence methods Serum type Cell typea Anti-HSV-i AntiHSV2 HSV-2 tumor- HSV-1 tumor- Weanling serum b serume bearing hamster bearing hamster hamster serumt serumd serume l + ± - ± - KOS-6-lh i HEF + HSV-2 strain 333J HEF + HSV-1 strain Patton" HEF' a Cells were grown on glass cover slips, air-dried, and fixed for 10 min in acetone at room temperature. Details of the indirect immunofluorescence technique are presented in Materials and Methods. The fixed cells were examined for specific cytoplasmic fluorescence. bprepared in weanling Syrian hamsters by three injections of UV-irradiated HSV-1 strain Patton. c Prepared in weanling Syrian hamsters by three injections of UV-irradiated HSV-2 strain 333. d From hamsters with tumors induced by the injection of HSV-2-transformed HEF cells ( , T#1). efrom hamsters with tumors induced by the injection of HSV-1 transformed HEF cells ( ). ' From 4- to 5-week-old normal weanling Syrian hamsters. g HEF cells transformed after infection with UV-irradiated HSV-1 strain h HEF cells transformed after infection with UV-irradiated HSV-1 strain KOS. HEF cells transformed after infection with UV-irradiated HSV-2 strain 333. Normal HEF cells infected with HSV-2 strain 333 (1 PFU/cell) for 24 h. k Normal HEF cells infected with HSV-1 strain Patton (1 PFU/cell) for 24 h. 'Normal HEF cells.

7 VOL. 12, 1973 HSV-1 TRANSFORMATION OF HAMSTER CELLS 215 particles or oncornavirus groupispecific (gs) antigens (21). It was therefore of interest to test the HSV-1-transformed cell lines for the presence of hamster (gs-1) and mammalian (gs-3) C-type particle antigens. HEF cells which had been transformed by the KOS and strains of HSV-1 were fixed for 10 min in acetone and tested for the presence of gs antigens by the indirect and direct immunofluorescence techniques. Antiserum specific against the gs-1 antigen was supplied by B. Hampar (National Institutes of Health, Bethesda, Md.), and antiserum against gs-3 antigen was supplied by R. E. Wilsnack (Huntingdon Research Center). No gs antigens were detected in either transformed cell line (Table 4). The absence of such markers is presumptive (though not conclusive) evidence that C-type particles played no role in the transforming event possibly induced by HSV-1. DISCUSSION The transformation of HEF cells after exposure to two strains of UV-irradiated HSV-1 was c 60 0 z cz SERUM / DILUTION FIG. 5. Neutralization of HSV-1-Patton (----) and HSV ( ) by sera from Syrian hamsters with tumors induced by the injection of cells into newborns. Sera from two hamsters with tumors induced by HSV-1-transformed cells (T#1, 0; and T#2, 0) were tested. Controls utilized a serum pool from hamsters immunized with UVirradiated HSV-1 (-). Normal hamster sera (not shown) did not neutralize either HSV-1 or HSV-2. TABLE 3. Detection of HSV-specific antigens in tumor cells with immunofluorescence reagents Serum typeb a ~~~HSV-2 HSV-1Wen Cell type0 Anti- Anti- tumor- tumor- lwean HSV-1 HSV-2 bearing bearing ling serum serum hamster hamster hs serum serumc serum T#Jd T# T# T#1e HEF cellsf a Cells were grown as in Table 2. bsera were prepared as in Table 2. c Sera from three different tumors were tested with identical results. dhef cells transformed by HSV-1 strain that had been grown in tissue culture for three passages after excision from a tumor-bearing hamster. T#1, T#2, and T#3 were from three animals which developed tumors after the injection of 2 x 105 cells from the in vitro transformed cell line ehef cells from a hamster tumor induced by the infection of cells transformed by UV-irradiated HSV-2 strain 333 ( T#1). ' Normal HEF cells. a logical extension of similar experiments which demonstrated the in vitro transforming potential of HSV-2. HSV-1 and HSV-2 are related viruses and share common antigens as well as common nucleotide sequences (1, 8, 15, 23). However, any attempts to implicate HSV-1 in human cancer by the epidemiological techniques that have been successful for HSV-2 would be extremely difficult because of the widespread occurrence of this virus in nature. An overwhelming percentage of adults have developed antibodies to HSV-1 before maturity, and an attempt to quantitate the possible role of HSV- 1 in human cancer by using the seroepidemiological methods which associated HSV-2 with cervical carcinoma would be extremely difficult. Therefore, results presented in this communication are the first indication that HSV-1 may have the potential to play an important role in cancer. Most experimental virus tumor systems deal with leukemias, lymphomas, or sarcomas which, although important, do not constitute the majority of human neoplasms. A model DNA virus transformation system concerned with carcinomas has not been available. It is therefore of potential importance that HSV- 1-transformed cells have an epithelial morphol-

8 216 DUFF AND RAPP J. VIROL. TABLE 4. Immunofluorescence tests for the presence of hamster leukosis virus antigens in HSV-1-transformed hamster cell linesa Trans- Anti Anti Wean- Cell type' forming HaLV-gs MLV-gs ling virus serumc serumd hamster serume KOS-6-1 HSV HSV HSV T#1 HSV T#2 HSV T#3 HSV T#1 HSV J20-NRK Murine sarcoma virus HDC-22 None HEF None a Transformed hamster embryo cells were tested for the presence of group-specific (gs) antigens by the direct immunofluorescence technique as described previously (21). 'Three cell lines were tested before passage in newborn hamsters (KOS-6-1, , and ). Four cell lines transformed by HSV-1 and HSV-2 ( T#1, T#2, T#3, and T#1) were tested after one passage in a newborn Syrian hamster. The J20-NRK cell line (cells transformed by a murine sarcoma virus) and HDC-22 (a transformed hamster cell line) were used for positive controls. Normal HEF cells were used as the negative control. Similar results with the and T#1 cells have been previously published (21). c Supplied by Berge Hampar (National Institutes of Health) and directed against the hamster-specific gs-1. d Supplied by R. E. Wilsnack, Huntingdon Research Center, and reacts with the gs-3 antigen associated with mammalian oncornavirus and oncornavirus-infected cells. e Collected from 4- to 5-week old Syrian hamsters. ogy and induce carcinomas upon injection into hamsters. It will be necessary to evaluate a large number of HSV-1-transformed cell lines to determine if there is a continuing relationship between HSV-1 and epithelial transformation. It will be difficult to determine the specific role of HSV-2 and HSV-1 in the induction and maintenance of the neoplastic state. However, several other laboratories have now demonstrated that HSV-2 can transform mouse (Munyon, personal communication), hamster (L. Kutinova', et al., Abstr. Seventh Meet. Eur. Tumour Virus Group, p. 19, 1972), and human cells (3). The oncogenic transformation of HEF cells by HSV-1 is additional confirmation of the cancer potential of human herpes simplex viruses and extends previous observations made with mouse cells to which a virus-specified enzyme was hereditably transferred by HSV-1 (17). ACKNOWLEDGMENTS We wish to thank Myron Katz, Doreen Lakatosh, Patricia Hafler, and Dennis Kresge for outstanding technical assistance. This study was conducted under Public Health Service contract no within the Special Virus Cancer Program of the National Cancer Institute. LITERATURE CITED 1. Bronson, D. L., B. J. Graham, and H. Ludwig Studies on the relatedness of herpes viruses through DNA-RNA hybridization. Biochim. Biophys. Acta 259: Churchill, A. E., and P. M. Biggs Agent of Marek's disease in tissue culture. Nature (London) 215: Darai, G., and K. Munk Human embryonic lung cells abortively infected with herpes virus hominis type 2 show some properties of cell transformation. Nature N. Biol. 241: Duff, R., and F. Rapp Oncogenic transformation of hamster cells after exposure to herpes simplex virus type 2. Nature N. Biol. 233: Duff, R., and F. Rapp Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultraviolet-irradiated herpes simplex virus type 2. J. Virol. 8: Duff, R., and F. Rapp The induction of oncogenic potential by herpes simplex virus. In Persistent virus infections, p The Gustav Stern Symposium on Perspectives in Virology VIII. Academic Press Inc., New York. 7. Epstein, M. A., B. G. Achong, and Y. M. Barr Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 1: Geder, L., and G. R. B. Skinner Differentiation between type 1 and type 2 strains of herpes simplex virus by an indirect immunofluorescent technique. J. Gen. Virol. 12: Glaser, R., R. G. Duff, and F. Rapp Ultrastructural characterization of hamster cells transformed following exposure to ultraviolet-irradiated herpes simplex virus type 2. Cancer Res. 32: Henle, G., and W. Henle Immunofluorescence in cells derived from Burkitt's lymphoma. J. Bacteriol. 91: Henle, G., W. Henle, and V. Diehl Relation of Burkitt's tumor-associated herpes type virus to infectious mononucleosis. Proc. Nat. Acad. Sci. U.S.A. 59: Henle, W., V. Diehl, G. Kohn, H. zur Hausen, and G. Henle Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157: Huebner, R. J., and G. J. Todaro Oncogene of' RNA tumor viruses as determinants of cancer. Proc. Nat. Acad. Sci. U.S.A. 64: Hunt, R. D., L. V. Mel6ndez, N. W. King, C. E. Gilmore, M. D. Daniel, M. E. Williamson, and T. C. Jones Morphology of a disease with features of malignant lymphoma in marmosets and owl monkeys inoculated with Herpesuirus saimiri. J. Nat. Cancer Inst. 44: Kieff, E., B. Hoyer, S. Bachenheimer, and B. Roizman Genetic relatedness of type 1 and type 2 herpes simplex virus. J. Virol. 9: Mel6ndez, L. V., M. D. Daniel, R. D. Hunt, and F. G. Garcia An apparently new herpesvirus from

9 VOL. 12, 1973 HSV-1 TRANSFORMATION OF HAMSTER CELLS 217 primary kidney cultures of the squirrel monkey (Saimiri sclureus). Lab. Anim. Care 18: Munyon, W., E. Kraiselburd, D. Davis, and J. Mann Transfer of thymidine kinase to thymidine kinaseless L cells by infection with ultraviolet-irradiated herpes simplex viruses. J. Virol. 7: Naib, Z. M., A. J. Nahmias, W. E. Josey, and J. H. Kramer Genital herpetic infection: association with cervical dysplasia and carcinoma. Cancer 23: Nazerian, K., J. J. Solomon, R. L. Witter, and B. R. Burmester Studies on the etiology of Marek's disease. II. Finding of a herpesvirus in cell culture. Proc. Soc. Exp. Biol. Med. 127: Rapp, F Variants of herpes simplex virus: isolation, characterization, and factors influencing plaque formation. J. Bacteriol. 86: Rapp, F., R. Conner, R. Glaser, and R. Duff Absence of leukosis virus markers in hamster cells transformed by herpes simplex virus type 2. J. Virol. 9: Rawls, W. E., W. A. F. Tompkins, and J. L. Melnick The association of herpesvirus type 2 and carcinoma of the uterine cervix. Amer. J. Epidemiol. 89: Schneweis, K. E., and A. J. Nahmias Antigens of herpes simplex virus type 1 and 2-immunodiffusion and inhibition passive hemagglutination studies. Z. Immunitaetsforsch. Allergie Klin. Immunol. 141: Wolfe, L. G., L. A. Falk, and F. Deinhardt Oncogenicity of Herpesvirus saimiri in marmoset monkeys. J. Nat. Cancer Inst. 47: