Interferon In Vitro. serum. However, for experimental purposes, Eagle. minimal essential medium containing 2% fetal bovine

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1 INFECTION AND IMMUNrrY, Nov. 1975, p Copyright C) 1975 American Society for Microbiology Vol. 12, No. 5 Printed in U.S.A. Murine Cytomegalovirus: Induction of and Sensitivity to Interferon In Vitro HERBERT K. OIE,* JOHN M. EASTON, DHARAM V. ABLASHI, AND SAMUEL BARON Cancer Institute* and National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Received for publication 28 May 1975 Moderate amounts of viral inhibitor were produced by mouse embryo (ME) cultures infected with two strains of plaque-purified murine cytomegalovirus (MCV). This inhibitor was shown to be interferon, based on the possession of similar properties. The growth studies of MCV in ME cells showed that interferon was produced as early as 4 h after infection, infectious virus was produced between 12 to 16 h, and cytopathic effect was produced between 16 to 18 h. Since MCV-induced interferon production and the subsequent development of antiviral state occurred early, the long eclipse period may be due to an interferonmediated delay of virus replication. Pretreatment of ME cells with varying concentrations of interferon before infection with MCV did not result in increased interferon production, but at high pretreatment doses a slight inhibitory effect on interferon production was observed. In vitro sensitivity studies showed that small doses of MCV were highly sensitive to the antiviral action of interferon, but higher viral doses proved to be markedly resistant. Although the available evidence does not permit a definitive interpretation of the mechanism by which MCV may show differing sensitivities to interferon action, the presence of a small interferon-resistant fraction of virus-infected cells may account for the observations. Murine and human cytomegaloviruses (CV) generally induce little or undetectable amounts of interferon (15, 16, 18, 28, 34). Actually, they may even actively inhibit interferon production. In addition, these viruses are reported to be relatively resistant to the antiviral action of interferon (16, 28). The present study was undertaken to reevaluate the role of interferon during MCV infection in vitro using an improved interferon assay and high-titered interferon preparations. The results indicated that (i) the sensitivity of CV in cell culture is highly dependent on the challenge dose of CV used in the assay, (ii) the strains of MCV used in the present study induced moderate amounts of interferon in mouse embryo (ME) cells, and (iii) interferon was detectable in the medium of infected mouse cultures 8 to 12 h before infectious virus. MATERIALS AND METHODS Cell cultures. Secondary ME cultures were prepared from primary cells, derived from 14- to 16-dayold NIH Swiss embryos. The growth medium for ME cells consisted of Eagle reinforced medium (2) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. The experimental medium was the same, except the serum content was reduced to 2% (REM2). Mouse L cells were cultured in suspension in Eagle spinner medium plus 10% fetal bovine serum. However, for experimental purposes, Eagle minimal essential medium containing 2% fetal bovine serum was used. All experiments were conducted with cells grown at 37 C in tubes (16 by 150 mm) positioned vertically in a humidified CO2 incubator. Viruses. A derivative of the Smith strain of MCV, CV201-3, was obtained from L. M. Martos, Flow Laboratories, Inc., Rockville, Md. Upon detecting polyoma virus in the CV201-3 pool, two substrains (MCV-P1 and MCV-P2) were isolated from plaques picked at end point dilutions and cell-free virus pools were prepared in secondary ME cells (1). MCV-P2, which was used in most of the experiments, was checked and found to be free of polyoma and other common murine virus contaminants by the MAP test (9) (kindly performed by M. J. Collins, Jr., and J. C. Parker, Microbiological Associates, Inc., Bethesda, Md.). The pools of MCV-P1 and MCV-P2 titered and plaque-forming units (PFU) per ml, respectively, when plaque assayed in secondary ME cultures maintained in fluid medium. GD-VII virus was produced and plaqued in BHK-21 cells (106 9 PFU/ml). Sindbis virus was prepared and plaque titrated in chicken embryo monolayer cultures (108 2 PFU/ml). Vesicular stomatitis virus (VSV), Indiana strain, was grown in chicken embryo cells but plaqued in mouse L cells (107-5 PFU/ml). GD-VII virus, Sindbis virus, and VSV were plaqued under an agar overlay medium. Preparation of interferon. Mouse serum interferon was produced by intravenous injection of Newcastle disease virus (NDV) into 25-g Swiss mice as 1012

2 VOL. 12, 1975 INTERFERON AND CYTOMEGALOVIRUS 1013 previously described (3). Its titer was U/ml. Preparation of MCV-induced viral inhibitor. Secondary ME cultures were infected with MCV at an input multiplicity of infection (MOI) of 1 to 5. After an adsorption period of 2 h at 37 C, the cultures were washed twice with Earle balanced salt solution, fed with REM2, and incubated at 37 C until the cytopathic effect (CPE) had involved 75 to 100% of the cell sheet. The medium was harvested, ph 2-treated overnight at 4 C, readjusted to ph 7, and clarified by centrifugation at 1,500 rpm for 10 min. Interferon assay. The antiviral activities of MSIand MCV-induced viral inhibitor were determined by the GD-VII hemagglutinin yield reduction method in mouse L cells (27). In every assay performed, a standard laboratory mouse serum interferon containing U/ml was titrated in parallel. The standard reference mouse serum interferon (no. G ) also titered U/ml by this assay. RESULTS Production and detection of viral inhibitor. Antiviral activity was initially demonstrated in fluids from ME cultures infected with CV201-3 (the strain contaminated with polyoma virus), but the titers were consistently low (Table 1). In contrast, viral inhibitor production by ME cells infected with the polyoma-free substrains, MCV-P1 and MCV-P2, was significantly greater (P < 0.01 and P < 0.005, respectively). Due to the differences reported in the detection of interferon production by MCV-infected cell cultures, an additional effort was made to confirm the requirement of MCV infection for the production of the viral inhibitor by secondary ME cells. Aliquots of MCV-P2 or CV201-3 were mixed with either normal mouse serum or sufficient mouse anti-mcv serum (prepared against a polyoma virus-free MCV pool) to completely neutralize virus infectivity when incubated for 1 h at 37 C. Fresh secondary ME cultures were infected with neutralized MCV and normal serum-treated MCV, as well as the untreated virus. As controls, secondary ME cultures were exposed to medium in place of virus. When the cultures exposed to infectious virus showed 75 to 100% CPE, the fluids from all cultures were harvested and assayed for antiviral activity. At the time the fluids were harvested, CPE in cultures infected with normal serum-treated MCV was comparable to that seen in cultures infected with virus not exposed to serum. A set of cultures infected with antibody-neutralized virus showed no CPE 7 days postinfection. The results (Table 2) showed that only cultures infected with virus capable of producing CPE (untreated and normal serumtreated MCV) were able to induce the production of the viral inhibitor. Fluids from cultures TABLE 1. Antiviral activities in culture fluids from MCV-infected ME cells Inhibitor titer (U/mi) MCV strain Expt 1 Expt 2 Expt 3 Expt 4 CV MCV-P MCV-P TABLE 2. Effect ofantiserum neutralization ofmcv infectivity on viral inhibitor production Medium from: Inhibitor titer (U/mi) CV201-3 MCV-P2 Normal ME culture 0 0 ME culture infected with MCV ME culture infected with MCV, treated with normal mouse serum ME culture infected with 0 0 MCV, neutralized with mouse anti-mcv serum exposed to medium only or antibody-neutralized virus contained no antiviral activity. Characterization of the viral inhibitor. Some of the physical, chemical, and biological properties of the viral inhibitor produced by MCV-infected secondary ME cultures were determined. It was stable at ph 2 for 24 h at 4 C; the antiviral activity was cell bound and could not be removed by repeated washings; and it was not sedimented at 38,000 rpm for 3.5 h. Exposure to 0.2 mg of trypsin per ml for 1 h at 37 C completely destroyed the antiviral activity, indicating the protein nature of the inhibitor. Species specificity was shown by the protection of mouse L and secondary ME cultures but not chicken, rabbit, or human cells. Fifty- to 100-fold less antiviral action was observed in rat embryo cells. Finally, antiviral action was demonstrated against VSV, GD-VII virus, and Sindbis virus, showing that the action of the inhibitor affected several unrelated viruses. These properties are consistent with those of interferon (25). Time course of virus replication and interferon production. Tube cultures of secondary ME cells were infected with MCV-P2 at an MOI of approximately 5 in a volume of 0.2 ml. After an adsorption period of 2 h at 37 C, during which time the tubes were rotated at frequent intervals to redistribute the virus, the cultures were washed three times with Earle balanced salt solution, fed with REM2, and incubated at

3 1014 OIE ET AL. 37 C. At predetermined time interval. s (see Fig. virus synthesis leveled off, coinciding with max- 1), three cultures were observed for CPE and imal (90 to 100%) CPE. Interferon, however, then frozen at -20 C until all samiples were was detectable in the fluids from MCV-infected taken. The cultures were then thawe4d and the cultures as early as 4 h after infection. Produc- pooled, tion increased until about 36 to 48 h, when samples for each time interval werre sonically treated (15 s, 20 kc/s, 4 C), and centri- maximal titers (2.0 to 2.6 log10 U/ml) were at- tained. fuged (2,000 rpm, 10 min). The supernatant fluids were divided into two aliquots. One was MCV-induced interferon production by used to determine virus production Iby plaque ME cells pretreated with interferon. Experiassay; the other was assayed for inte,rferon. ments were done to determine whether pretreat- Viral CPE was first observed 16 to 1, 8 h postin- ment of ME cells with interferon affected subse- cells quent interferon production induced by MCV. fection and consisted of a few individual appearing swollen and rounded. At 2A4 h, 25 to ME cells were exposed to interferon concentra- 50% of the cell monolayer showed CPE. In tions ranging from 10 to 1,000 U/culture for 24 h many areas, the cells which had enliarged and before infection with either MCV-P2 (MOI = 5) rounded up had formed large aggrega tes. Giant or with Sindbis virus (MOI = 10). Sindbis virus cells were also observed in areas of CPE. By 48 challenge was used to determine the resist- Virus ance developed after exposure to the various h, 90 to 100% of the cells were involvred. production was initially detected 12 to 16 h after concentrations of interferon. These cultures infection (Fig. 1), slightly earlier thain the first were harvested 24 h after infection and virus observation of CPE. Thereafter, the a roduction yields were determined by the hemagglutinawas essentially linear until 36 to 4E5 h, when tion assay using gander erythrocytes (8). Resistance is expressed as the log10 reduction in hemagglutinin yields in interferon-treated cultures 7.0 _ 6.0 as compared to untreated controls. MCV-infected cultures were harvested 48 h postinfection when controls (cultures not treated with interferon) showed 75 to 90% CPE. The cultures 5.0 were scored for CPE before harvesting of fluids w C,) zdi I 5.0 z 0 LLI D 4.0 _ a. U 0 >~~~ L I0 o INFECT. IMMUN. I for interferon assay. m Pretreatment of ME cells with interferon did m not result in increased interferon production _4. 0 (priming effect) at any concentration of inter- Z feron used (Table 3). However, at 100 U per < culture or more, a slight but probably insignifim cant reduction of interferon yields was ob- - served. In addition, at interferon pretreatment _30 o levels (640 to 1,000 U/culture) which resulted _ ) in complete protection against Sindbis virusinduced CPE, changes suggestive of MCV-inz duced CPE were observed, suggesting that MCV was less sensitive than Sindbis virus to -X the action of interferon. m In vitro sensitivity of MCV to interferon. K ME cells were incubated overnight with 10 or '0 100 U of interferon per culture and then in- 1.0 fected with varying doses (10 to 10,000 PFU/culture) of 2.0~ MCV-P2. Four replicate cultures were used for each combination of interferon concentration and virus dose. Before and _ 0 after virus absorption (2 h at 37 C), the cells were washed twice with Earle balanced salt HOURS POST INFECTION solution, fed with interferon-containing meand inter- dium, incubated at 37 C in a humidified CO2 FIG. 1. Time course of infectious virus feron production by ME cells infected with MCV. (*) incubator, and examined daily for CPE develop- Logio PFU/ml assayed in ME cells. (0) Interferon ment. To maintain the desired interferon contiters assayed in mouse L cells by the GD-VTII hemag- centrations in the medium throughout the duraglutinin reduction assay. Curves represenit averages tion of the experiment, the culture fluids were of data from two experiments. replaced with fresh interferon-containing me-

4 VOL. 12, 1975 INTERFERON AND CYTOMEGALOVIRUS 1015 dium every 3rd day. When the cells that had not received interferon showed 75 to 100% CPE, the cultures were frozen at -20 C until virus yields were determined by plaque assay in secondary ME cells. The results showed that interferon-induced resistance to MCV-P2 was inversely related to the virus dose (Table 4). At low viral input, significant yield reductions were demonstrated; however, as the dose was increased, even continuous treatment with 100 U of interferon was not able to protect the cells or reduce virus yields significantly. Based on the results in Table 4, the titer of the interferon preparation used in this experiment was 1,600 U/ml when an MCV challenge dose of 10 PFU/culture was used; however, if an MCV dose of 10,000 PFU was used, the anti- TABLE 3. Interferon production by interferonpretreated ME cells infected with MCV Pretreatment Resistance to Sindbis vi- Interferon Pdoseeoefinmteer- rus (log,ore- % MCV-in- yield after feron feron (U/ml) duction in duced CPE MCV infec- HA tion (log,,,) (U/mi) viral yield)a , a HA, Hemagglutinin. TABLE 4. Effect ofvirus challenge dose on sensitivity to interferon Interferon (log1o Virus challenge dose ducytione U/culture) (log,o PFU/culture) (log10) 1.0 MCV: Sindbis: b 2.0 MCV: Sindbis: a MCV yield reduction is expressed as the difference in log10 PFU between interferon-treated and untreated cultures. b For comparison of sensitivities of MCV and an interferon-sensitive virus, Sindbis HA yield reduction data were transferred from Table 4. viral activity would be <16 U/ml. In contrast, interferon titers obtained by using small (multiple growth cycles) or large (single growth cycles) challenge doses of another virus (VSV) were not found to be significantly different (17). Similarly, in the present study a Sindbis virus challenge dose of PFU was markedly inhibited (Table 4). DISCUSSION Although some investigators have reported the detection of interferon production during CV infection (15, 18, 34), others have not (16, 28). In the present study, two plaque-purified strains of MCV were found to induce moderate amounts of interferon in ME cultures. This inhibitor was shown to be interferon based on the possession of similar properties. Moreover, this interferon was produced as a result of MCV infection because (i) antiviral activity could be demonstrated only in fluids from MCV-infected cultures, and (ii) neutralization ofvirus infectivity with specific anti-mcv serum rendered the virus noninfectious and incapable of inducing interferon production. The results reported here confirm the findings of Henson and Smith (18) but do not provide an explanation as to why others (28) have failed to detect interferon in MCV-infected culture fluids. One possibility may be the presence of an undetected agent (e.g., polyoma virus) in the stock MCV preparation which might interfere with MCV-induced interferon production, as was probably the case early in this study. Another possibility may be the presence of interferon antagonists in the preparations tested for antiviral activity (6, 7, 12, 13, 14, 31, 33). Still another possibility could be the use of an interferon assay of low sensitivity İnterferon synthesis in ME cells began very early (4 h) after MCV infection, yet maximum titers were not reached until 36 to 48 h. In contrast, interferon was not detected until 7 to 8 h postinfection in mouse L cells infected with the Roakin strain of NDV (21) and 10 to 12 h after infection of ME cells with the Herts strain of NDV (Oie, unpublished data). In both cases, NDV-induced interferon production peaked within 24 h. Thus, MCV-induced interferon production began earlier but peaked at a much later time than NDV-induced production. MCV growth in ME cells was characterized by a long eclipse period; new infectious virus was not detected until 12 to 16 h after infection. CPE was observed 4 to 6 h later. VSV challenge during the eclipse period (first 12 h postinfection) showed that the cells had developed sufficient resistance to reduce VSV yields by 10- to

5 1016 OIE ET AL. INFECT. IMMUN. 16-fold (Oie, unpublished data). This low-grade resistance could delay MCV replication sufficiently to account for the 12-h eclipse period. A final test of this hypothesis must await development of methods of suppressing the interferon system during MCV infection. Inhibition of virus replication by treating ME cells with interferon before infection with MCV did not result in increased interferon production (priming effect), but at high pretreatment doses a modest reduction in MCV-induced interferon yields was observed. A similar but more pronounced state of refractoriness to interferon production was reported for mouse L cells treated with interferon preceding induction with NDV (Herts strain) (26). Several investigators have reported that the CV were not sensitive to interferon action (16, 28). Rabson et al. (29), however, showed that the in vitro replication of human CV could be inhibited provided that sufficient quantities of interferon and an interferon-sensitive cell system were used. Our results with murine CV confirm the findings of these investigators. When high concentrations of interferon were used against low doses of MCV, significant inhibition of virus yields was observed. However, even high concentrations of interferon were not measurably effective against large challenge doses of MCV. Other investigators (22), using a plaque reduction assay, found that cell-associated CV was highly resistant to the antiviral action of interferon, whereas cell-free CV was very sensitive. They suggested that the resistance of the cell-associated virus might be due to the production of antagonists to interferon by the infected cells. In a study not reported here, we had found that ME cells infected with MCV did not produce interferon antagonists. Protection of the intact animal by interferon is highly dependent on the virus challenge dose (4, 5). In contrast, most investigators have found that the titer of an interferon sample assayed in cell cultures was independent of the dose of virus used (10, 17, 20, 23, 24). In two early reports, however, a minimal decrease in interferon sensitivity was observed in vitro as the virus challenge dose (Sindbis virus and M6 virus) was increased (11, 19, 30). More recently a strong dose dependency was reported for herpes simplex virus inhibition by rabbit interferon (35). A virus challenge dose dependency was also observed in repeated experiments in which interferon-treated human cells were infected with the AD-169 strain of human CV (J. M. Easton, unpublished observations). Similarly, in the present study, small doses of murine CV were found to be sensitive to interferon action, whereas large doses were highly resistant. Thus, CV and herpes simplex virus are the only clear examples of viruses which manifest strong virus dose dependency to interferon action in vitro. It would be of interest to know whether other members of the herpesvirus group manifest similar virus dose dependency to interferon action. A possible interpretation is based on the occurrence of a small fraction (0.1 to 15%) of virusinfected cells which are relatively resistant to the antiviral action of interferon (32). Very small infecting doses of virus would have a low probability of establishing interferon-resistant infected cells, whereas large doses of virus would have a high probability of establishing such resistant, infected,cells. A partial test of this hypothesis would be an in vitro study correlating the effect of virus challenge dose on interferon sensitivity with the magnitude of the resistant fraction in different virus-cell systems. ACKNOWLEDGMENTS We thank Agnes H. Simmons and Sara L. Myers for excellent technical assistance. LITERATURE CITED 1. Ablashi, D. V., L. M. Martos, R. V. Gilden, and B. Hampar Preparation of rabbit immune serum with neutralizing activity against a simian cytomegalovirus (SA6). J. Immunol. 102: Bablanian, R., H. J. Eggers, and I. Tamm Studies on the mechanism of poliovirus-induced cell damage. I. The relationship between poliovirus-induced metabolic and morphological alterations in cultured cells. Virology 26: Baron, S., and C. E. Buckler Circulating interferon in mice after intravenous injection of virus. Science 141: Baron, S., C. E. Buckler, R. M. Friedman, and R. V. McCloskey Role of interferon during viremia. II. Protective action of circulating interferon. J. Immunol. 96: Catalano, L. W., Jr., and S. Baron Protection against herpes virus and encephalomycarditis virus encephalitis with double-stranded RNA inducer of interferon. Proc. Soc. Exp. Biol. Med. 133: Chany, C., and C. Brailovsky Stimulating interaction between viruses (stimulons). Proc. Natl. Acad. Sci. U.S.A. 57: Chany, C., and F. Robbe-Maridor Enhancing effect of the murine sarcoma virus (MSV) on the replication of the mouse hepatitis virus (MHV) in vitro. Proc. Soc. Exp. Biol. Med. 131: Clarke, D. H., and J. Casals Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7: Collins, M. J., Jr., and J. C. Parker Murine virus contaminants of leukemia viruses in transplantable tumors. J. Natl. Cancer Inst. 49: Finter, N. B Quantitative haemadsorption, a new assay technique. I. Assay of interferon. Virology 24: Finter, N. B Interferon assays and standards, p.

6 VOL. 12, 1975 INTERFERON AND CYTOMEGALOVIRUS In N. B. Finter (ed.), Interferons. North- Holland Publishing Company, Amsterdam. 12. Fournier, F., S. Rousset, and C. Chany Investigations on a tissue antagonist of interferon (TAI). Proc. Soc. Exp. Biol. Med. 132: Ghendon, Y. Z On the ability of certain viruses to block the effect of interferon. Acta Virol. (Engl. ed.) 9: Ghendon, Y. Z., I. G. Balandin, and L. M. Babushkina On the mechanism of the ability of certain viruses to block the action of interferon. Acta Virol. (Engl. ed.) 10: Glasgow, L. A Cytomegalovirus interference in vitro. Infect. Immun. 9: Glasgow, L. A., J. B. Hanshaw, T. C. Merigan, and J. K. Petralli Interferon and cytomegalovirus in vivo and in vitro. Proc. Soc. Exp. Biol. Med. 125: Hallum, J. V., and J. S. Youngner Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures. J. Bacteriol. 92: Henson, D., and R. D. Smith Interferon production in vitro by cells infected with the murine salivary gland virus. Proc. Soc. Exp. Biol. Med. 117: Ho, M Kinetic consideration of the inhibitory action of an interferon produced in chick cultures infected with Sindbis virus. Virology 17: Isaacs, A Action of interferon on the growth of sublethally irradiated virus. Virology 9: Lancz, G. J., and T. C. Johnson Temporal relationship between virus and interferon biosynthesis in L cells infected with Newcastle disease virus. Proc. Soc. Exp. Biol. Med. 132: Lang, D. J., M. Thomas, and I. Gresser Protection par l'interferon de cellules embryonnaires humaines contres l'infection par le virus cytomegalique. C. R. Acad. Sci. Ser. D 268: Lindenmann, J., and G. E. Gifford Studies on vaccinia virus plaque formation and its inhibition by interferon. III. A simplified plaque inhibition assay of interferon. Virology 19: Lockart, R. J., Jr., and T. Sreevalsan The effect of interferon on the synthesis of viral nucleic acid, p In Viruses, nucleic acids, and cancer. Williams and Wilkins, Baltimore. 25. Lockart, R. J., Jr Biological properties of interferon; criteria for acceptance of a viral inhibitor as an interferon, p In N. B. Finter (ed.), Interferons. North-Holland Publishing Company, Amsterdam. 26. Margolis, S. A., H. Oie, and H. B. Levy The effect of interferon, interferon inducers or interferon induced resistance on subsequent interferon production. J. Gen. Virol. 15: Oie, H. K., C. E. Buckler, C. P., Uhlendorf, D. A. Hill, and S. Baron Improved assays for a variety of interferons. Proc. Soc. Exp. Biol. Med. 140: Osborn, J. E., and D. N. Medearis, Jr Studies of relationship between mouse cytomegalovirus and interferon. Proc. Soc. Exp. Biol. Med. 121: Rabson, A. S., S. A. Tyrrell, and H. Levy Inhibition of human cytomegalovirus in vitro by doublestranded polyribocytidylic-inosinic acid (Poly I,rCr). Proc. Soc. Exp. Biol. Med. 131: Sellers, R. F., and M. Fitzpatrick An assay of interferon produced in rhesus monkey and calf kidney tissue cultures using bovine enterovirus M6 as challenge. Br. J. Exp. Pathol. 43: Sheaff, E. T., and R. B. Stewart A substance enhancing virus growth and antagonistic to interferon action. Can. J. Microbiol. 14: Takemoto, K. K., and S. Baron Non-heritable interferon resistance in a fraction of virus populations. Proc. Soc. Exp. Biol. Med. 121: Truden, J. L., M. M. Sigel, and L. S. Dietrich An interferon antagonist: its effect on interferon action in Mengo-infected Ehrlich ascites tumor cells. Virology 33: Vaczi, L., E. Horvath, and Gy. Hadhazy. 1965/1966. Studies on the conditions of interferon production by cells infected with herpes viruses. Acta Microbiol. Acad. Sci. Hung. 12: Weissenbacher, M., M. A. Galin, E. Chowchuvech, N. Schachter, and S. Baron Protection by polyinosinic polycytidilic acid complex of rabbit eye tissue cultures infected with herpes simplex virus. Effect of neomycin and virus challenge dose. Invest. Opthalmol. 9: