Ultracentrifugal Inoculation

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1 INFECTION AND IMMUNITY, Oct. 1980, p /80/ /05$02.00/0 Vol. 30, No. 1 Mechanisms of Herpes Simplex Virus Infectivity Enhanced by Ultracentrifugal Inoculation RICHARD B. TENSER* AND MARIE E. DUNSTAN Departments of Microbiology and Medicine (Neurology) and Specialized Cancer Research Center, The Pennsylvania State University, College ofmedicine, Hershey, Pennsylvania Ultracentrifugation of very dilute suspensions of herpes simplex virus directly onto monolayer cells grown in centrifuge tubes was studied. Enhanced infectivity by ultracentrifugation was similar at 40C and at 35 to 370C. The high infectivity levels of cultures centrifuged at 40C were further examined by infectious center assays. At 40C, the numbers of infectious centers in control (noncentrifuged) cultures were almost 100-fold fewer than in control cultures at 370C. However, the numbers of infectious centers in cultures ultracentrifuged at 40C were similar to those ultracentrifuged at 370C. The great difference in the numbers of infectious centers between 4 and 370C control cultures, in contrast to the similarity between 4 and 370C ultracentrifuged cultures, indicated that ultracentrifugation at 40C enhanced infectivity possibly by facilitation of herpes simplex virus penetration into monolayer cells. We previously reported that ultracentrifugation of herpes simplex virus (HSV) directly onto monolayer cells grown in centrifuge tubes markedly enhanced the detection of HSV (7). Monolayer cells tolerated ultracentrifugation well, and infectivity was increased almost 100-fold when ultracentrifugal inoculation (UCIN) of very dilute HSV suspensions was employed. Enhanced infectivity probably resulted from a concentrating effect; that is, concentrating the sparsely distributed HSV particles onto the monolayer cells by centrifugal force. Other mechanisms, however, such as enhanced infectivity of individual virus particles due to some effect of the centrifugal force on the particles or on the monolayer cells, could not be ruled out. We now report results of HSV UCIN studies indicating one such additional mechanism of enhanced infectivity. Variation of the temperature at which UCIN was performed was studied, and enhancement of infectivity at 4 C was surprisingly similar to that at 370C. By utilizing infectious center assays of monolayer cells after UCIN, it appeared that at a low temperature, UCIN may enhance the infectivity of HSV particles independently of the concentrating effect, possibly by facilitating virus penetration into cells. MATERIALS AND METHODS Virus. HSV type 1 strain KOS was grown in primary rabbit kidney cells by standard methods (8). The infectivity titer of stock virus was 5 x 10' plaqueforming units (PFU) per ml (log tissue culture infective dose [TCIDro] 7.2). For ultracentrifugation studies, virus was diluted 10-fold to limiting dilutions in 193 tris(hydroxymethyl)aminomethane (Tris) buffer (0.02 M, ph 7.3) or in Eagle minimal essential medium (MEM). Growth of cells for ultracentrifugation. Vero cells (Flow Laboratories, Rockville, Md.) were grown in sterile centrifuge tubes in medium 199 supplemented with sodium bicarbonate, antibiotics, and 10% fetal calf serum. Monolayer cultures were grown in Oakridge-style polycarbonate tubes (25.3 by 89.6 mm; Sorvall, Newtown, Conn.) and were incubated at about a 450 angle as described previously (7). Ultracentrifugation. Centrifuge tubes containing Vero cell monolayer cultures were inoculated with 25 ml of dilute HSV suspension and were placed in a fixed-angle Beckman 60 Ti rotor so that cells would receive the maximal centrifugal force. In this way, suspended material would be pelleted onto the monolayers. Four tube cultures were used for each dilution. Ultracentrifugation (Sorvall OTD-2) at 20,000 rpm (28,000 x g) for 2.3 h was performed as described previously (7). Centrifugation was performed at 4 C, 20 to 22 C, or 35 to 37 C; control (uncentrifuged) tube cultures were similarly inoculated and incubated at similar temperatures and at a similar angle. After centrifugation or control inoculation, virus-containing medium was discarded, and 2 to 3 ml of maintenance medium (medium 199 supplemented with sodium bicarbonate, antibiotics, and 5% fetal calf serum) was added. In some experiments, UCIN and control cultures were incubated at 37 C for 1 h after the centrifugation period before the virus inoculum was decanted. Infectivity determinations. The TCID!,/25 ml was calculated by the method of Reed and Muench (6) for centrifuged and control tube cultures. TCID5r values were also calculated for tube cultures in which infectious centers were determined after ultracentrifugation. TCIDro values for these tube cultures were determined by assuming that if at least one infectious center were present, cytopathology would be evident.

2 194 TENSER AND DUNSTAN IC assay. After ultracentrifugation, the number of infected cells in centrifuged and control tube cultures was determined by infectious center (IC) assays. The medium was decanted, and monolayer cells were trypsinized and suspended in growth medium. Suspended Vero cells were diluted 10-fold and were seeded onto confluent rabbit kidney monolayer cultures in 60-mm plastic culture dishes. After adsorption for 1 h at 370C in a 5% humidified CO2 atmosphere, the cultures were overlaid with 0.5% methylcellulose and incubated for 4 days. At that time, the methylcellulose was removed, cultures were stained with 0.1% neutral red in Tris, and IC were determined by counting plaques. In addition to determining the number of IC by trypsinization of cultures immediately after ultracentrifugation, IC assays were performed on centrifuged and control cultures incubated for 1 h at 370C after ultracentrifugation. The experimental procedure utilized is shown in Fig. 1. RESULTS Effect of medium and temperature on UCIN. Enhanced infectivity with UCIN was observed when centrifugation was performed at 4CC, 20 to 220C, and 35 to 37CC (Table 1). Somewhat surprisingly there was no significant difference in TCID50 infectivity titer at the three temperature conditions. The mean TCIDso titer for all of the Tris control groups was 7.6; the corresponding titer for the UCIN groups was 9.3. In the Eagle MEM groups, the values were 7.8 and 9.0, respectively. Since there was no significant difference in the Tris groups or in the 20 to 220C and the 35 to 370C Eagle MEM groups whether or not serum (10%) was present (data not shown), the values for these groups stock INFECT. IMMUN. were pooled. However, for the 40C Eagle MEM groups, if serum was not present, the cultures severely deteriorated during the 2.3-h inoculation period; therefore, only infectivity titers obtained with serum present were determined. UCIN of HSV in Tris was consistently, although only slightly, superior to UCIN in Eagle MEM. However, monolayer cultures also appeared to be more intact after UCIN in Tris, and this diluent was therefore used in subsequent experiments. IC determinations. To quantitate results obtained by the UCIN procedure other than by determining TCID50, we determined the numbers of infected monolayer cells (IC) after inoculation with dilute HSV suspensions. Dilute HSV was used to decrease the probability that infected cells were infected by more than one virus particle. The results seem valid since 10- fold virus dilutions resulted in ca. 10-fold IC differences (Table 2). After determining the number of IC present per 106 monolayer cells with several HSV concentrations, we calculated the IC titers (Table 2). The latter calculation was analogous to determining the PFU titer of stock virus from the number of PFU present in dilutions of the stock. There was a 25-fold difference in IC titers between the control and UCIN groups (Table 2). In addition, on a qualitative basis, IC were detectable after UCIN with HSV that was 100-fold more dilute than that which produced detectable IC after control inoculation. From Table 2 it appears that calculating the IC titer was a valid way to measure infectivity. no centrifugation ultra- no ultratrifugation centifugation cen centrifugation TC ID50 11 / \ no \\ no TCID50 incubation incubation incubation incubation f i i IC IC IC IC FIG. 1. Experimental design. Stock HSV was highly diluted, and 25-ml portions were inoculated into centrifuge tubes containing Vero cell monolayer cultures. Inoculation of control and ultracentrifuged cultures was performed at several temperatures. For IC assays, weprocessed the cultures immediately after inoculation or after an intervening 1-h incubation at 370C. TCID5o was calculated for cultures in which IC were determined as described in the text.

3 VOL. 30, 1980 TABLE 1. ULTRACENTRIFUGAL INOCULATION OF HSV 195 Effect of medium and temperature during centrifugation on enhanced infectivity of HSV by UCINO Infectivity titer, log TCIDro (mean ± standard error) Medium Control, no centrifugation UCIN 40C 20 to 220C 35 to 37"C 40C 20 to 22"C 35 to 370C Tris 7.3 ± ± ± ± ± ± 0.12 Eagle b 7.8 ± ± ± 0.29b 8.9 ± ± 0.18 MEM All cultures were infected with 25 ml of dilute HSV suspension. b Eagle MEM for these groups contained 10% serum. Differences between UCIN and control groups were significant at the P < level. TABLE 2. Number of IC per 106 cells and IC titers of control and UCIN cultures inoculated at 35 to 370C IC/106 cells after inoculation Culture with HSV (PFU/ml)a IC titerb Control x UCIN x 10' ± 0.8^ a Tube cultures were inoculated with 25 ml of the dilute HSV suspensions. The results were mean values of three experiments. b IC titers were calculated from the number of IC/106 cells in cultures inoculated with several dilutions of HSV. Since the stock HSV used had an infectivity titer of 105 PFU/ml as determined in RK plates, 100 PFU/ml corresponded to a 106 dilution. 'Differences between control and UCIN IC titers were significant at the P < level. This was substantiated by directly comparing IC titer with TCID50 in both control and UCIN cultures and those inoculated at 40C and at 35 to 370C. The IC titer was correlated with log TCID5o over 4 log units, although the IC titer was almost 10-fold lower than the TCID5o titer (Fig. 2). Results from control and UCIN cultures inoculated at 4 and 370C are shown. Since IC titer calculation appeared to be a satisfactory means of determining infectivity, this method was further used to investigate enhanced infectivity by UCIN. When IC titers of UCIN and control cultures kept at 35 to 370C during the period of centrifugation were compared to those from similar cultures that had an additional 1-h incubation period at 370C after centrifugation, no significant difference was seen (Fig. 3). IC infectivity was enhanced by UCIN compared to control cultures; however, the additional 1-h incubation period had no effect. In control cultures infected at 40C, the 1-h incubation period at 370C significantly increased the IC titer. Not surprisingly, incubation at 370C before trypsinization increased the IC titer in the 40C control cultures 6) C-) c0 0 C Log TCID50 FIG. 2. Logarithmic plot of IC titers and log TCID5o. Results were obtained with control and UCIN cultures at 40C and at 35 to 37 C. to values similar to those in the 370C control groups. Most importantly, post-ucin incuba- tion at 370C had only a minimal effect on tube cultures ultracentrifuged at 40C. That is, incubation at 370C before trypsinization was not necessary for the UCIN cultures centrifuged at 40C; whereas it was necessary for the 40C control cultures. It appears that in the UCIN cultures kept at 40C, HSV infectivity was protected from trypsinization and it is probable that HSV penetration into the monolayer cells was facilitated per se. DISCUSSION A concentrating effect with ultracentrifugation would be predicted based on the calculated

4 196 TENSER AND DUNSTAN U 10) Control UCIN FIG. 3. Logarithmic plot of IC titers for control and UCIN cultures. Monolayer cells in centrifuge tube cultures were trypsinized immediately after inoculation at 4 C or at 35 to 37 C or after an additional 1-h incubation at 37 C. Means and standard errors are for three experiments. clearing of HSV from suspension using the equation: T = K/S (1) where T equals time in hours to sedimentation; K equals the clearing factor of the rotor; and S equals the sedimentation coefficient of HSV. Since the rotor was operated at less than its maximal speed of 60,000 rpm, at which speed the K factor of 63 would apply, the actual K factor (Ka) was calculated from the equation: K. = K (N,/Na)2 (2) where Ka equals the clearing factor at the speed used; N, equals the maximal rotor revolutions per minute; and Na equals the actual revolutions per minute used. From equation 2 the appropriate K factor is calculated as 567. When this value is used for K in equation 1 and when the S value for HSV is estimated as 500 to 600, T is calculated as ca. 1 h (0.9 to 1.1 h). As would be expected, enhanced infectivity with UCIN was not noted when the HSV suspension to be tested was first clarified by ultracentrifugation at 28,000 x g for 2.3 h (unpublished data). For most of the UCIN experiments, 25 ml of diluted virus was inoculated into each tube culture. As discussed previously (7), the large volume was necessary to ensure that some infec- INFECT. IMMUN. tious virus was present. Such assurance would be impossible using 0.1-ml portions of highly dilute virus suspensions, unless large numbers of samples were tested. With large volumes, although the number of PFU per centrifuge tube was controlled, the HSV PFU present were more correctly indicated as a viral concentration than as an amount. Since HSV concentration was an experimental variable, infectivity enhancement by UCIN has been dicussed as a concentrating, rather than as a concentration effect. Enhanced infectivity not resulting from a concentrating effect was in part examined by temperature experiments. It has been reported that HSV infectivity is markedly decreased when inoculation is performed at 40C (2). We observed, however, that the increased TCID,% of cultures centrifuged at 40C was similar to those centrifuged at 20 to 220C and at 35 to 370C. This raised the possibility that enhanced infectivity using UCIN was due to more than a concentrating effect. The importance of inoculation temperature was even more evident in the IC experiments. IC titers were more than 10-fold greater for the 370C control cultures and for 40C control cultures with a post-inoculation incubation period at 370C than for 40C cultures without the incubation period. Significantly, after UCIN at 40C, infectivity was similar to that of the 37'C UCIN cultures; post-inoculation, incubation at 370C was not necessary. It appears that UCIN at 40C enhanced infectivity by permitting establishment of infection otherwise not possible. This is clearly seen by the minimal 370C control versus UCIN IC titer difference and the almost 100-fold difference between the 4'C control versus UCIN IC titer. Ultracentrifugation of infectious virus onto monolayer cells was described previously, although mechanisms were not discussed (1, 5). In studies of low-speed (800 to 1,900 x g) centrifugation-enhanced infectivity with mouse cytomegalovirus, Osborn and Walker (4) and Hudson et al. (3) attributed increased infectivity to enhanced adsorption of virus onto monolayers, an inherent property of the virus. They reported that enhanced HSV infectivity, although much less than with cytomegalovirus, was probably the result of other mechanisms. We observed intermediate levels of enhanced HSV infectivity using low-speed (100 to 3,000 x g) centrifugation (7), and in conjunction with the results obtained with high-speed centrifugation, centrifugal infectivity enhancement appeared to be due, at least in part, to a concentrating effect. Virus infectivity enhancement by UCIN as a result of the concentrating effect may be of value in the isolation of sparsely distributed infectious

5 VOL. 30, 1980 virus particles. It is also apparent, however, that while enhanced HSV infectivity with UCIN is due in part to a concentrating effect, that at least in some situations, infectivity may be increased by a mechanism independent of this effect, possibly by enhanced virus penetration into cells. ACKNOWLEDGMENTS This work was supported in part by Public Health Service Teacher Investigator Award NS00248 from the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS), grant NS14568 from the NINCDS, and grant CA from the National Cancer Institute. We thank Fred Rapp for helpful discussions, Melissa Reese for editorial assistance, and Kelly Yingst for secretarial assistance. ULTRACENTRIFUGAL INOCULATION OF HSV 197 LITERATURE CITED 1. Gey, G. O., F. B. Bang, and M. K. Gey Responses of a variety of normal and malignant cells to continuous cultivation and some practical problems of these responses to problems in the biology of disease. Ann. N.Y. Acad. Sci. 58: Huang, A. S., and R. R. Wagner Penetration of herpes simplex virus into human epidermoid cells. Proc. Soc. Exp. Biol. Med. 116: Hudson, J. B., V. Misra, and T. R. Mosmann Cytomegalovirus infectivity: analysis of the phenomenon of centrifugal enhancement of infectivity. Virology 72: Osborn, J. E., and D. L. Walker Enhancement of infectivity of murine cytomegalovirus in vitro by centrifugal inoculation. J. Virol. 2: Padgett, B. L., and D. L. Walker Use of centrifugal force to promote adsorption of myxoma virus to cell monolayers. Proc. Soc. Exp. Biol. Med. 111: Reed, L. J., and H. Muench A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27: Tenser, R. B Ultracentrifugal inoculation of herpes simplex virus. Infect. Immun. 21: Tenser, R. B., and M. Dunstan Herpes simplex virus thymidine kinase expression in infection of the trigeminal ganglion. Virology 99: Downloaded from on May 8, 2018 by guest