Establishment of a Nonproductive Herpes Simplex Virus

Transcription

1 INFECTION AND IMMUNrrY, July 1975, p, Copyright American Society for Microbiology Vol. 12, No. 1. Printed in U.SA. Establishment of a Nonproductive Herpes Simplex Virus Infection in Rabbit Kidney Cells J. J. KELLEHER,* J. VARANI, AND W. W. NELSON Microbiology Department, University of North Dakota School of Medicine, Grand Forks, North Dakota Received for publication 17 April 1975 A nonproductive infection of rabbit kidney cells was established with a type 2 strain of herpes simplex virus after incubation of the virus-infected cells at 41 C. Although infectious virus could not be recovered from cells disrupted by freeze-thawing or sonication after incubation at 41 C, spontaneous reactivation of virus growth occurred in 84% of the cultures after lag periods of variable length when cultures of viable cells were transferred to 37 C and incubated. Forty-one percent of the cultures had lag periods of 4 or more days, and 24% had lag periods of 7 or more days. The longest lag period was 45 days. A similar infection was established in WI-38 cells, but attempts to establish the infection in human kidney cells was not successful. This nonproductive infection is being used to study the effects of various physical and chemical agents on herpes simplex virus in a latent state. Infection with herpes simplex virus (HSV) normally results in the establishment of a latent infection after the initial contact between the virus and host (23). During the latent infection the virus may survive in the sensory ganglion cells. The activation of virus growth after surgical manipulation of the trigeminal nerve supports this hypothesis (2, 3). Experimental latent HSV infections have been established in sensory ganglion cells of mice (24) and rabbits (25). The virus, or at least the virus genome, may also survive in nonneural cells. Royston and Aurelian (22) detected HSV-specific antigens in cervical epithelial cells from women with cervical carcinoma. Furthermore, HSV was recovered from a culture of human cervical cells after spontaneous reactivation of virus growth occurred in cefs in vitro (1). Attempts have been made to establish latent HSV infections in vitro. A number of carrier culture infections have been established (4, 6, 9-11, 14, 15, 26, 27). Production of infectious virus, though not completely eliminated, is reduced to a very low level in this type of infection. O'Neill et al. (17) established a cell culture system in which it appeared that infectious HSV was completely eliminated from the cells after treatment with cytosine arabinoside. Although infectious virus could not be recovered from the infected cells during treatment, virus growth resumed following a lag period after removal of the inhibitor from the culture medium. Longson (13) and Ratcliffe (19) reported that type 2 strains of HSV failed to replicate at temperatures above 40 C in RK,3 cells or in Vero cells. Oh and Schlenke (16) noted that type 2 strains produced only focal cell destruction in rabbit kidney (RK) cells at 40 C but that virus multiplication with concomitant cell destruction increased upon further incubation of the cultures at 37 C. Yoshino et al. (28) reported that growth of the HF strain of HSV was greatly suppressed in chicken embryo cells at 40 C but that normal multiplication resumed after the cultures were transferred to 35 C. We have found that multiplication of a type 2 strain of HSV was completely inhibited in RK cells at 41 C and that virus growth did not immediately resume in many of the cultures after transfer to 37 C. With further incubation of these cultures at 37 C, spontaneous reactivation of virus 128 growth did occur in a number of the cultures after lag periods of up to 45 days. During the period when virus growth was not occurring at 37 C, virus could not be recovered from the infected cells after disruption of the cells by freeze-thawing or sonication. This report describes the establishment and partial characterization of this nonproductive infection. This model infection is being used to study the effects of chemical agents on HSV in a nonproductive state. MATERIALS AND METHODS Cells. RK cells were prepared according to a procedure outlined by Youngner (29) for the preparation of monkey kidney cells. Cells were propagated in human kidney growth medium (12) containing 100 U of penicillin, 100 gg of streptomycin, and 0.25 jig of

2 VOL. 12, 1975 Fungizone per ml. When full monolayers were reached, the growth medium was replaced with maintenance medium. The maintenance medium consisted of Eagle minimal essential medium with 2% fetal calf serum and antibiotics. Wistar-38 (WI-38) cells were purchased from Grand Island Biological Co. (Grand Island, N. Y.) in tubes. Upon arrival in the laboratory, the cell culture medium was replaced with our maintenance medium. Human kidney (HK) cells were purchased from Microbiological Associates (Bethesda, Md.) and handled in the same way as the WI-38 cells. Virus. HSV type 2, strain MS (ATCC-VR540), was purchased from the American Type Culture Collection, Rockville, Md., and used throughout these studies. The virus was originally isolated from the brain of a 50-year-old female patient with multiple sclerosis (8) and has been passed several times in RK cells since isolation. For these studies stock cultures of virus were prepared in RK cells. Virus quantitation was done using a plaque assay method described by Dolan et al. (5). Tenfold dilutions of virus were made in Hanks balanced salt solution. Duplicate flasks of RK cells were inoculated with 0.2 ml of each dilution and incubated at 37 C for 4 days under a semisolid overlay, which consisted of Eagle minimal essential medium with 2% fetal calf serum, antibiotics and 2% methylcellulose (400 centipoise). After 4 days, the semisolid overlay was decanted, and the cells were fixed for 10 min with anhydrous methanol and strained for 20 min with Giemsa strain. Plaques were counted both macroscopically and microscopically. A modification of the mean tissue culture infective dose assay was used to assay virus in viable cells. The cell monolayers to be assayed for virus were dispersed into individual cells by treatment with 0.25% trypsin. The dispersed cells were centrifuged at 150 x g for 10 min and resuspended in human kidney growth mediujn. Tenfold dilutions of the cells were made in growth medium. Cells from each dilution were added to full monolayers of RK cells, which served as substrates upon which the added cells could attach and which also served as efficient indicator cells which readily showed cytopathogenic effect (CPE) once active virus growth occurred. When CPE was observed in infected cultures, the virus was passed once in RK cells and identified as HSV on the basis of neutralization tests. Neutralization of 102 plaque-forming units (PFU) of virus by 1:64 dilution of commercial anti-hsv serum (Microbiological Associates) was accepted as evidence that the virus was HSV. High-temperature incubation. A water jacketregulated incubator was used to incubate cells at 41 C. The temperature varied not more than ±0.5 degrees. The temperature was set at 41.5 C so that the minimum temperature would not fall below 41.0 C. Cell viability staining. Cells incubated at 37 C and 41 C were monitored for viability by the erythrocin B vital staining procedure (18). After dispersal of the monolayers into individual cells with 0.25% trypsin, the cells were centrifuged and resuspended in Hanks balanced salt solution. To each 1 ml of suspended cells was added 0.2 ml of a 0.4% erythrocin B solution (Fisher Scientific Co., Fairlawn, N.J.). After incubation for 5 min at room temperature, the NONPRODUCTIVE HSV INFECTION IN RK CELLS 129 cells were counted in a hemocytometer. Viable cells remained unstained, whereas nonviable cells stained pink. Immunofluorescence studies. Virus-specific antigens were detected by indirect immunofluorescence staining. Antiserum to HSV was prepared in New Zealand white rabbits by repeated subcutaneous injections of HSV-infected RK cells in Freund complete adjuvant. Serum prepared in this way was able to neutralize 102 PFU of virus at a 1:40 dilution in a standard neutralization test. Fluorescein isothiocyanate-conjugated serum prepared in sheep against rabbit globulin was purchased from Grand Island Biological Co. Cells were grown on glass.cover slips in Leighton tubes. Prior to staining, the monolayers were washed three times in phosphate-buffered saline and fixed in acetone for 30 min at - 20 C. After fixation in acetone, the cells were again washed three times in phosphate-buffered saline and then stained sequentially with the anti-hsv serum and the anti-rabbit globulin serum. Staining was done according to the method of Ross et al. (21). Both the anti-hsv serum and the anti-rabbit globulin serum were diluted 1:4 in phosphate-buffered saline prior to use. After staining, the cover slips were mounted on clean glass slides using a 90% glycerol-10% phosphate-buffered saline solution for mounting. Stained preparations were viewed by dark field microscopy under ultraviolet light using a Leitz ortholux microscope. A UG1 excitation filter and K430 barrier filter combination was used. Acridine orange studies. Acridine orange staining was done following a procedure by Gluck and Kulovich (7). Cells grown on cover slips in Leighton tubes were washed three times in phosphate-buffered saline and fixed for 10 min in Carnoy fixative. The monolayers were stained immediately after fixation or allowed to remain in the fixative for up to 5 days. The monolayers were mounted on clean glass slides after staining and viewed by dark field microscopy under ultraviolet light. Microscopy was done with the Leitz ortholux microscope. A BG12 excitation filter and K530 barrier filter were used. RESULTS Inhibition of virus growth at 41 C; recovery of virus at 37 C after extended lag periods. Cultures of RK cells were infected with HSV at a low multiplicity of infection (MOI) of (500 PFU of virus per culture containing 106 cells), incubated at 41 C, and observed daily for CPE. No CPE was seen at any time during 5 days of observation. On each day duplicate cultures were freeze-thawed three times and assayed for infectious virus in RK cells. No virus was recovered from any of the cultures. In addition to freeze-thawing, 40 cultures were also treated by sonication at 10,000 cycles/s for 30 s. No virus was recovered from any culture incubated at 41 C by this method of cell disruption as well. A study was done to determine how stable the

3 130 KELLEHER ET AL. virus was at 41 C. The rate of inactivation of virus in the presence of viable RK cells and nonviable RK cells is shown in Fig. 1. The nonviable RK cells were obtained by freezethawing cultures of viable RK cells. Virus was inactivated at a logarithmic rate. In the presence of viable RK cells the half-life of the virus was approximately 100 min. In the presence of nonviable cells the half-life was only 36 min. No virus was recovered from any culture that was incubated at 41 C for longer than 12 h. Cultures infected with 5 x 103 to 5 x 104 PFU of virus (MOI of to 0.05) were incubated at 41 C for 1 to 7 days and then transferred to 37 C and incubated at this temperature. Although virus could not be recovered from cells incubated at 41 C when the cells were freeze-thawed or sonicated, virus was not irreversibly inactivated at 41 C and virus growth did occur when viable cells were transferred to 37 C (Table 1). CPE was observed in 86 cultures out of 200 within 3 days after the cultures were transferred to 37 C. In an additional 82 cultures, CPE was observed only after delays ranging from 4 to 45 days. The period of time during which no 4 E 3 -J: Time (hours) FIG. 1. Thermal inactivation of HSVat 41 C in the presence of viable and nonviable RK cells. Cultures of viable RK cells and freeze-thawed RK cells were infected with virus and incubated at 41 C. At 2-h intervals duplicate cultures were freeze-thawed three times and assayed for virus in RK cells. Symbols: Viable cells, (A); nonviable cells, (-). INFECT. IMMUN. TABLE 1. Recovery of HSVat 37 C after incubation at 41 C in RK cells No. of cultures in which virus was No. of cultures recovered after incubation at 37 C incubated at 41 Ca or days days dmaye 34 (1-2 days) (3-7 days) a Cultures were infected with between 5 x 103 to 5 x 104 PFU of virus. evidence of virus growth was seen in the cultures at 37 C was defined as the lag period. Thirty-four cultures had lag periods of 4 to 6 days, and 48 cultures had lag periods in excess of 6 days. The longest lag period observed was 45 days, and the average lag period was 15.3 days. Overall, virus was eventually recovered from 168 of the 200 cultures initially infected for a recovery rate of 84%. Cultures in which virus growth did not occur after transfer to 37 C were routinely discarded after 60 days, although some cultures were maintained for up to 150 days. The cells were kept in a viable condition during the long holding periods by replacing the culture medium with fresh maintenance medium every 4 days. Examination of the supernatant fluids for HSV always yielded no virus during the lag periods. Once CPE was observed, however, the virus spread rapidly and destroyed the culture within 2 to 3 days. High titers of infectious HSV were recovered from cultures at this time. Virus was transferred to cultures of uninfected RK cells to confirm the presence of virus, and neutralization tests with commercial anti-hsv serum confirmed that the virus isolated from these cultures was HSV. Estimation of the amount of virus surviving incubation at 41 C. Two methods were used to estimate the amount of virus that was able to survive incubation at 41 C. One method involved use of the viable cell assay described in Materials and Methods. RK cultures were infected with 105 PFU of virus (MOI of 0.1) and incubated at 41 C. After incubation for 2 to 6 days the monolayers were dispersed into individual cells, and specified numbers of cells were added to carrier cultures of RK cells. The carrier cultures were then incubated at 37 C and monitored for CPE (Table 2). Virus was recovered from 12 of 12 cultures that had received 105 cells, from 8 of 12 cultures that had received 10' cells, and from 7 of 12 cultures that had received 103 cells. Virus was not recovered from cultures that had received 102 or 101 cells from 41 C incubation.

4 VOL. 12, 1975 In another experiment cultures were infected with between 4 and 4,000 PFU of virus (MOI of to 0.004) and incubated at 41 C for 2, 4, or 6 days. After incubation at 41 C, cultures were transferred to 37 C and continued incubating at this temperature. Cultures at 37 C were monitored daily for CPE (Table 3). Virus was recovered from 19 of 24 cultures that had received 4,000 PFU of virus and from 15 of 24 cultures that had received 400 PFU of virus. Virus was only recovered from three cultures that had received 40 PFU and from no cultures that had received 4 PFU of virus. These studies indicate that as much as 1% of the inoculum virus survived incubation at 41 C in some of the cultures. Effect of 41 C preincubation on RK cell viability and ability to support replication of exogenous virus at 37 C. The effect of 41 C incubation on the survival of RK cells was studied using the erythrocin B vital staining procedure. Cultures were incubated at 37 C and 41 C. On four consecutive days duplicate cultures from both temperatures were stained with erythrocin B, and an estimate of the percentage of viable cells in each culture was determined. There was no significant difference in cell viability between 41 C and 37 C. The percentage of viable cells in cultures incubated at 41 C was 76 i 6%. The percentage of viable cells at 37 C was TABLE 2. Recovery of HSVin carrier cultures at 37 C after addition of cells from cultures infected at 41 C to the carrier cultures Length of incubation period at 41 C (days) No. of carrier cultures in which virus was recovered after addition of the following numbers of cells:" 10' ' /4 3/4 3/4 0/4 0/4 4 4/4 2/4 3/4 0/4 0/4 6 4/4 3/4 1/4 0/4 0/4 a One out of 10 cells was infected as determined by control studies in which cultures were infected with virus, incubated at 37 C for 6 h, and treated in exactly the same manner as the experimental cultures. TABLE 3. Amount of virus needed to establish a latent infection Length of incubation period at 41 C (days) No. of cultures in which virus was recovered at 37 C after incubation at 41 C with the following amount. of virus (PFU/culture): 4, /8 7/8 2/8 0/8 4 6/8 5/8 0/8 0/8 6 6/8 3/8 1/8 0/8 NONPRODUCTIVE HSV INFECTION IN RK CELLS ± 6%. At both temperatures the percentage of viable cells remained constant over the 4-day period. Cells preincubated at 41 C were tested for their ability to support virus replication after transfer to 37 C. Two parameters of virus growth were measured: (i) the ability of virus to adsorb to cells, and (ii) the ability of cells to produce infectious virus. The amount of virus able to adsorb to cells was determined by adding 0.2 ml of a stock culture of HSV to cells that had been preincubated at either 37 C or 41 C. After adsorption periods of 2 or 4 h at 37 C, the supernatant fluids were decanted and the cells were washed and overlayed with the semisolid overlay. Cultures were then incubated for 4 days at 37 C, and plaques were counted at this time. The amount of virus that adsorbed to cells preincubated at 41 C was comparable to the amount that adsorbed to cells preincubated at 37 C (Table 4). The ability of cells preincubated at 41 C to produce infectious virus at 37 C was measured by infecting cultures preincubated at 41 C with 500 PFU of virus (MOI of ) and incubating them at 37 C. Cultures preincubated at 37 C served as controls. At 48 and 72 h cultures were freeze-thawed and assayed for virus. The amount of virus produced by cultures preincubated at 41 C was similar to the amount produced by cultures preincubated at 37 C (Table 5). Immunofluorescence staining. Indirect fluorescent antibody staining was done to determine if viral antigens were produced at 41 C. Leighton tube cultures of RK cells were infected TABLE 4. Adsorption of HSV to RK cells preincubated at 37 and 41 C for 2 days Amt of virus which Preincubation Adsorption afsorbe toice temp (C) period (h) adsorbed to cells TABLE 5. Production of HSV at 37 C in RK cells preincubated at 37 and 41 C for 2 days Time after Amt of virus treincubation infection produced temp (C) (h) (PFU/ml) x x x 10" x 106

5 132 KELLEHER ET AL. with 105 PFU of virus (MOI of 0.1) and incubated at 37 C and 41 C. Specific fluorescence could be seen as early as 4 h after incubation at 37 C. Fluorescence was located in the perinuclear area and obliterated the appearance of the nuclear membrane. By 6 to 8 h after infection the nucleus was no longer distinct from the rest of the cell and the cell was a mass of bright fluorescence. Most of the cells incubated at 41 C looked identical to uninfected control cells. There were isolated cells, however, which showed the perinuclear fluorescence. Isolated fluorescing cells were seen in cultures incubated at 41 C for 1, 2, 3, or 4 days. Acridine orange staining. Leighton tube cultures of RK cells were infected with 105 PFU of virus (MOI of 0.1) and incubated at 41 C. At 2-h intervals, cultures were removed from incubation, stained with acridine orange, and examined for morphological alterations. Cultures incubated at 37 C for 2 or 4 h looked identical to control cultures. Morphological alterations became visible in the nuclei of infected cells 6 to 8 h after infection. Infected cells showed a bright ring of fluorescence at the periphery of the nuclei accompanied by a decrease in the fluorescence in the center of the nuclei. In addition, the overall intensity of the staining increased in the infected cells. The nuclei of infected cells stained yellow, in contrast to the green staining of nuclei from uninfected cells. Cultures incubated at 41 C from 2 h to 4 days appeared identical to uninfected control cultures. There was no evidence of morphological alterations in any cell from these cultures. Establishment of the latent infection in WI-38 cells; failure to establish the infection in HK cells. Attempts were made to establish the latent infection in both WI-38 and HK cells. The first step was to determine if virus growth was blocked at 41 C in these cells. Tube cultures of WI-38 and HK cells were infected with 5 x 102 to 5 x 103 PFU of virus (MOI of to 0.005) and incubated at 41 C. On the following 3 days duplicate cultures of cells were freezethawed and assayed for virus in RK cells. No virus was recovered from any culture. Cultures of WI-38 and HK cells were infected with 5 x 108 to 5 x 10' PFU of virus (MOI of to 0.05), incubated at 41 C for 1 to 6 days, and then transferred to 37 C. Virus growth resumed in 59 out of 121 cultures of WI-38 cells originally infected for an overall recovery rate of 49% (Table 6). In 37 of these cultures there were lag periods of 4 or more days, and 16 cultures had lag periods of 7 or more days. The longest lag period was 53 days, and the average lag period of the last 16 cultures was 26.8 days. TABLE 6. Recovery of HSVat 37 C after incubation at 41 C in WI-38 and HK cells No. of cultures in which virus No. of was recovered after incubation Cell cultures at 37 C type incubated at 41 Ca or days days dmayse WI HK a Cultures were infected with between 5 x 103 to 5 X 104 PFU of virus. The infection in WI-38 cells was similar to the infection in RK cells. In contrast to this, the latent infection was not established in HK cells (Table 6). After transfer of virus-infected cultures from 41 C to 37 C, virus was recovered in only 8 of 62 cultures (13%). Only four cultures had lag periods in excess of 3 days, and none had lag periods of 7 or more days. DISCUSSION INFECT. IMMUN. Yoshino et al. (28) and Oh and Schlenke (16) reported that HSV failed to replicate normally at 40 C in chicken embryo fibroblasts and in RK cells and that active virus replication resumed when the cultures were transferred to 35 or 37 C. In these two studies virus growth was not completely eliminated at 40 C but only reduced to a very low level. When cultures were transferred to permissive temperatures, active virus growth resumed immediately. We have observed that a type 2 strain of HSV did not replicate in RK cells at 41 C. In this study virus growth was completely blocked and infectious virus could not be recovered after disruption of the cells. Furthermore, we observed that active virus growth did not resume immediately in a large number of cultures after they were transferred to 37 C. In 82 of 200 cultures there was a delay of 4 or more days before evidence of virus growth was detected after transfer to 37 C. Forty-eight of these cultures had lag periods of 7 or more days. The longest lag period observed was 45 days, and the average of these last 48 cultures was 15.3 days. When virus was inoculated directly onto cells at 37 C in control studies, evidence of virus growth could always be seen within 3 days irrespective of virus concentration. The establishment of the nonproductive infection was cell dependent. WI-38 cells responded in a manner similar to RK cells. The

6 VOL. 12, 1975 NONPRODUCTIVE HSV INFECTION IN RK CELLS 133 overall recovery rate of virus from WI-38 cells was 49%. Thirty-one percent of the cultures had lag periods of 4 or more days and 13% had lag periods of 7 or more days. The recovery rate of virus from HK cells was only 13%, and no culture had a lag period of 7 or more days. The reason for the low recovery rate from HK cells is not known. A latent infection can be defined as the situation which exists when virus is contained in a cell in which virus multiplication has been reversibly interrupted (20). During the period at 37 C before active virus growth resumed, the virus was latent. We have not studied the mechanism leading to the establishment of the latent state after preincubation at 41 C. We *have, however, used the latency attained by such manipulation to study the effects of various physical and chemical agents on HSV in a nonproductive state and feel that this is a useful model system for such studies. ACKNOWLEDGMENTS These studies were supported in part by Public Health Service Research Training Grant 5T 01 GM to J. Varani from the National Institute of General Medical Sciences and by grant 5 S01 RR05407 from the Division of Research Resources. LITERATURE CITED 1. Aurelian, L., J. D Strandberg, L. V. Melendez, and L. A. Johnson Herpesvirus type 2 isolated from cervical tumor cells grown in culture. Science 174: Carton, C. A Effect of previous sensory loss on the appearance of herpes simplex virus following trigeminal root section. J. Neurosurg. 10: Carton, C. A., and E. D. Kilbourne Activation of latent herpes simplex virus following trigeminal root section. New Engl. J. Med. 246: Coleman, V., and E. Jawetz A persistent herpes simplex virus infection in antibody-free cell culture. Virology 13: Dolan, T. M., J. D. Fenters, P. A. Fordyce, and J. C. Holper Rhinovirus plaque formation in WI-38 cells with methycellulose overlay. Appl. Microbiol. 16: Fernandez, C. 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