Assay of Variola Virus by the Fluorescent Cell-Counting Technique

Transcription

1 APPLIED MICROBIOLOGY, NOV., 1965 Copyright 1965 American Society for Microbiology Vol. 13, No. 6 Printed in U.S.A. Assay of Variola Virus by the Fluorescent Cell-Counting Technique NICHOLAS HAHON U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland Received for publication 6 May 1965 ABSTRACT HAHON, NICHOLAS (Fort Detrick, Frederick, Md.). Assay of variola virus by the fluorescent cell-counting technique. Appl. Microbiol. 13: A quantitative assay for infective variola virus particles was developed which is based on the enumeration of cells containing fluorescent viral antigen after infection of McCoy cell monolayers. The direct fluorescent-antibody technique was employed to stain cells. The efficiency of virus adsorption was markedly enhanced by centrifugation of virus inoculum onto McCoy cell monolayers at 5 X g for 15 min. By this procedure, a proportionality was obtained between the number of fluorescent cells and volume of inoculum. Observations on the sequential development of viral antigen within cells and counts of fluorescent cells showed that the optimal time for enumerating fluorescent cells was after an incubation period of 16 to 2 hr. A linear function existed between virus concentration and cell-infecting units. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The assay was demonstrated to be highly sensitive, precise, and reproducible. Since the introduction of the fluorescent cellcounting technique for the assay of Newcastle disease virus by Wheelock and Tamm (1961), the procedure has been applied successfully for the quantitative titration of adenovirus (Philipson, 1961), human cytomegalovirus (Goodheart and Jaross, 1963), and psittacosis virus (Hahon and Nakamura, 1964). In assays of these viruses, the technique has been shown to be sensitive, precise, reproducible, and rapid (results usually obtained in less than 24 hr). The applicability of the technique has been enlarged further with the development of a fluorescent cell-counting neutralization test to detect and to measure quantitatively psittacosis serum-neutralizing antibodies (Hahon and Cooke, 1965). In view of the marked advantages and potential of the fluorescent cellcounting technique in virus studies, the feasibility of using this procedure to assay poxviruses was investigated. This report describes the standardization of the fluorescent cell-counting technique for the quantitative assay of variola virus. MATERIALS AND METHODS Virus. The Yamada strain of variola virus was used throughout this study; its history has been described elsewhere (Hahon, Ratner, and Kozikowski, 1958). Virus suspension was prepared by inoculating the chorioallantoic membrane (CAM) of 11-day chick embryonated eggs with 5 X 13 pock-forming units of virus. After incubation at 35 C for 48 hr, infected membranes were harvested, made into a 5% suspension with phosphatebuffered saline (PBS), ph 7., and homogenized in a Waring Blendor. The suspension was partially purified by two cycles of differential centrifugation, 78 X g and 8, X g for 1 hr each. After the second cycle, the pellet was suspended in PBS, and the suspension was distributed in 1-ml portions into glass vials and stored at -6 C. The virus suspension assayed by CAM inoculation of chick embryonated eggs and enumeration of pock-forming units (Hahon et al., 1958) contained approximately 4 X 16 pock-forming units per milliliter. Cell line. The established cell line, McCoy, derived from human synovial tissue (Fernandes, 1959) was used in the fluorescent cell-counting for virus. Preliminary experiments with high multiplicities of virus to cells indicated that more than 99% of the cells were susceptible to infection by variola virus. Cell cultivation. Nutrient medium for the McCoy cell line consisted of mixture 199 containing.5% lactalbumin hydrolysate, 1% heat-inactivated calf serum, and 5,g of streptomycin and 75 j,g of kanamycin. Cells were maintained in mixture 199 and 5% calf serum. For virus assay, cells were cultivated on circular cover slips (15-mm diameter) inserted in flat-bottomed glass vials (18 by 1 mm). A 1-ml amount of cell suspension, containing 15 to 3 X 15 cells, was introduced onto cover slips that were then incubated at 35 C for 24 hr, or until a complete cell monolayer was 865 Downloaded from on September 25, 218 by guest

2 866 HAHON APPL. MICROBIOL. formed. Cover slip cell cultures were washed twice with 2 ml of maintenance medium prior to the addition of virus inoculum. Virus assay. Assays were usually carried out in duplicate or triplicate. Virus dilutions were prepared in maintenance medium and introduced in.2-ml volumes directly into vials containing cover slip cell cultures. Routinely, virus adsorption was carried out by centrifugation at 5 X g for 15 min at 23 to 25 C. For this procedure, vials were placed in slotted cups containing tube adapters, sealed with a screw-dome cover, and mounted on a four-place, pin-type head. Centrifugation was performed in an International centrifuge, size 2, model V. Cover slip cell cultures were rinsed twice with maintenance medium after adsorption of virus; 1 ml of the medium was added then to each vial. After incubation for 16 to 2 hr at 35 C, cover slips were rinsed twice with cold PBS, fixed with cold (-6 C) acetone, and either prepared immediately for immunofluorescent staining and cell counting or stored at -6 C for subsequent examination. In addition to the fluorescent cell-counting technique, virus was assayed by CAM inoculation of embryonated eggs (Hahon et al., 1958) and in roller-tube cell cultures (Hahon, 1958). In this study, the latter procedure was slightly altered; virus inoculum was adsorbed onto McCoy cell monolayers with tubes maintained in a stationary position at 35 C for 2 hr. After an incubation period of 6 days, the tissue culture 5% infective dose (TrcID5), based on cytopathic changes produced in cell monolayers by virus inoculum, was calculated by the method of Reed and Muench (1938). Variola antiserum conjugate and technique of immunofluorescent staining. For the preparation of antiserum, rabbits were immunized initially by multiple scarification with variola virus. When the cutaneous lesions had healed, rabbits were inoculated intraperitoneally with 1 ml of virus containing 5 X 17 pock-forming units in a series of four injections given at 2-week intervals. The animals were bled approximately 1 month after the last injection. Antiserum was conjugated with fluorescein isothiocyanate by the method of Riggs et al. (1958). The conjugate was adsorbed twice with mouse liver powder and used undiluted for staining. The direct fluorescent-antibody technique was employed to demonstrate immunofluorescence in infected cells. Fixed cell cultures were washed three times with PBS and stained with the conjugate for 3 min. The cover slip cell cultures were then rinsed in three changes of PBS to remove excess conjugate and were mounted in 1% glycerol in PBS. Fluorescence microscopy. Cover slip cell cultures were examined with an American Optical microscope equipped with a Fluorolume illuminator (model 645; Corning no. 584 and Schott BG-13 exciter filters, and an E. K. no. 24 barrier filter). Fluorescent cell-counting and calculations. At 43 times magnification with the optical system employed, 1,64 microscopic fields were contained in the area of a 15-mm cover slip. For each cover slip cell culture, 5 microscopic fields were examined for fluorescent cells. To calculate the number of cell-infecting units of virus per milliliter, the average number of fluorescent cells per field was multiplied by the number of fields per cover slip, the reciprocal of the dilution of virus inoculum, and a volume factor (for conversion to milliliters). RESULTS Virus adsorption. The applicability of centrifugal force for promoting adsorption of virus onto cell monolayers was investigated because previous studies with psittacosis virus confirmed the efficiency of centrifugation for this purpose (Hahon and Nakamura, 1964). An experiment was performed to determine the rate of variola virus adsorption onto cell monolayers during stationary incubation (35 C) and centrifugation (5 X g, 25 C). Because comparable fluorescent cell counts were obtained with the use of centrifugal force of 5 X g for 3 min or 1,25 X g for 15 min during virus adsorption, the former condition of centrifugation was selected for this test. To vials containing cover slip cell cultures,.2 ml of a 1-3 dilution of virus suspension was added. Vials were divided into two groups; each group was subjected to a different condition of virus adsorption. At designated intervals during the adsorption period, vials were removed, and cell cultures were then treated in the manner described for virus assay. The percentage of virus adsorbed during each interval with each procedure is shown in Fig. 1. Within 15 min, virus adsorption with the aid of centrifugation appeared to be complete, whereas approximately 6% was adsorbed during stationary incubation at 35 C for 2 hr. Since the efficiency and rapidity of virus adsorption onto cell monolayers by the use of centrifugal force was clearly superior to results obtained with stationary incubation, centrifugation at 5 X g for 15 min at room temperature was adopted as the standard procedure for virus adsorption. The efficiency of centrifugation for infecting cell monolayers from different volumes of inoculum is given in Table 1. The results revealed a proportionality between the number of fluorescent cells and the volume of inoculum. The rate of cell-virus contact appeared to be independent of the volume of inoculum employed in this experiment when virus adsorption was carried out with the aid of centrifugal force. Incubation period. As early as 6 hr after the introduction of viral inoculum, a faint fluorescent intracytoplasmic center was seen in a few cells. Downloaded from on September 25, 218 by guest

3 VOL. 13, 1965 ASSAY OF VARIOLA VIRUS 9 so 'o '7 > 6o z z 9- z MINUTES FIG. 1. Adsorption of variola virus onto cover slip cultures of McCoy cells by (A) centrifugal force (5 X g, 25 C) and (B) stationary incubation (35 C). TABLE 1. Proportionality between volume of inoculum and cell-infecting units of variola virus Volume Fluorescent cells CIU*/ml X 17 per 5 fields ml * Cell-infecting units of virus determined by fluorescent cell-counting technique in 2 hr. At 8 hr, foci of fluorescence could be distinguished readily at the periphery of the nucleus (Fig. 2). Examination of cell cultures at 16 and 2 hr revealed that the amount and intensity of fluorescence in cells had increased markedly. Fluorescence was diffused throughout the entire cell cytoplasm (Fig. 3), but none was discernible in the nucleus. Lengthy cytoplasmic extensions were common. Fluorescent cells could be easily enumerated during this period. At 24 hr, several rounded cells were present. Fluorescence in these and other cells was dense and granular in appearance at the periphery of the nucleus (Fig. 4). Rounded fluorescent cells were more numerous at 3 hr (Fig. 5), and various stages of constriction of cytoplasmic extensions were seen. At 48 hr, cytoplasmic extensions were generally absent. Rounded cells were abundant at that time, and their aggregation tended to form fluorescent foci. Occasionally, minute fluorescent extracellular particles were noted, and some cells had a fluorescent intracytoplasmic center that suggested a second cycle of virus infection. In general, the cell monolayer at 48 hr was disorganized. Counts of fluorescent cells were comparable between 16 and 24 hr after infection. The number of fluorescent cells was two- and threefold higher at 3 and 48 hr, respectively, than at 16 and 24 hr. That the fluorescence noted in cells was specific for viral antigen was confirmed by the serum-neutralization reaction. Fluorescent cells were sparse or absent when mixtures of virus and antiserum were inoculated onto cell monolayers; numerous fluorescent cells were noted when a mixture of virus and normal serum was used as inoculum. Based on the sequential development of viral antigen within infected cells and fluorescent cell counts, the optimal period for incubation of inoculated cell monolayers was established as 16 to 2 hr. Quantitative evaluations of the assay. Results in Fig. 6 reveal a linear relationship between twofold dilutions of virus over a range of 1.8 log units and the number of cell-infecting units of virus. These data suggest that each fluorescent cell was the consequence of infection by a single infective virus particle. In a single experiment, 1 determinations were made to estimate the precision of the fluorescent cell-counting procedure for variola virus. Cell cultures were infected with a standard quantity of virus inoculum and incubated in the prescribed manner. The number of cell-infecting units of virus per milliliter of inoculum ranged from 17 to 1.5 X 17, with a mean of 1.2 X 17. The standard deviation of.21 compared favorably with that obtained in studies with psittacosis virus (Hahon and Nakamura, 1964). The sensitivity of the cell-counting procedure was compared with that of two other methods for estimating the concentration of variola virus: enumeration of pock-forming units on the CAM of chick embryonated eggs and the cytopathic changes produced in roller-tube cultures as an index of virus infectivity. Table 2 shows that the sensitivity of the fluorescent cell-counting tech- Downloaded from on September 25, 218 by guest

4 868 HAHON APPL. MICROBIOL. Downloaded from FIG Fluorescent variola virus antigen in infected McCoy cell monolayers. FIG. 2. Minute areas of fluorescence seen 8 hr after infection. FIG. 3. Single cell exhibiting lengthy cytoplasmic extensions with diffuse fluorescence seen 16 hr after infection. FIG. 4. Numerous fluorescent cells seen 24 hr after infection. Note dense and granular appearance of fluorescent viral antigen. FIG. 5. Rounded fluorescent cells commonly seen 3 hr after infection. Two brightly fluorescent cells are in the center of the field. on September 25, 218 by guest

5 VOL. 13, 1965 ASSAY OF VARIOLA VIRUS 869 TABLE 3. Reproducibility of assay for variola virus by the fluorescent cell-counting technique o 14 x 12 a: 12 a.,a CL z zn6 I-,u4.64 FIG. 6. Linear function between the number of cell-infecting units and dilutions of variola virus. TABLE 2. -i I DILUTIONS OF VIRUS Comparison of different methods for the assay of variola virus Assay no. CIU*/ml PFUt/ml TCIDMot/ml Assay no. X 16 X 16 X Mean SD ±. 21 ± SE of mean * Cell-infecting units of virus determined by fluorescent cell-counting technique in 2 hr. t Pock-forming units of virus determined by CAM inoculation of chick embryonated eggs, 3- day incubation period. t Tissue culture 5% infective dose determined by cytopathic changes in roller-tube cultures of McCoy cells, 6-day incubation period. nique, within the limits of the doses employed, was superior to that of the other two procedures. In addition, less variation occurred among determinations by the former procedure. One other assay technique for variola virus was employed 64 Dilution of virus in inoculum Assay CI- - U*/ml no. 5.1 X 2.56 X 1.28 X 6.4 X 3.2 X XCIo* ' 1-' t Mean * Cell-infecting units of virus determined by fluorescent cell-counting technique in 2 hr. t Number of fluorescent cells per 5 fields X 12. WI 9r 4~ FIG. 7. Frequency distribution of fluorescent cells on monolayers of McCoy cells infected with variola virus. ---* Theorectical *-O Observed NUMBER OF FLUORESCENT CELLS PER MICROSCOPIC FIELD which is based on the enumeration of hyperplastic foci (Pirsch and Ptrlson, 1962). This assay gave an average titer of 6.8 X 16 focus-forming units per milliliter for three determinations. The reproducibility of the fluorescent cellcounting technique with the use of twofold dilutions of virus inoculum in each of four determinations is shown in Table 3. The results attest to the reproducibility of the assay at the concentration levels employed. The mode of distribution of fluorescent cells on a cover slip cell monolayer previously infected with virus after 2 hr of incubation was deter- 7 Downloaded from on September 25, 218 by guest

6 87 HAHON mined by examining 3 random microscopic fields. Observed frequencies of fluorescent cells corresponded closely to theoretical frequencies (Fig. 7). Since there was no significant departure from the theoretical Poisson distribution (X2 = with degree of freedom = 5), the distribution of fluorescent cells on cover slip cultures was random. DISCUSSION Although both the direct and indirect fluorescent-antibody techniques have been employed successfully for diagnostic purposes to demonstrate viral antigen in smears from smallpox lesions (Avakyan et al. 1961; Murray, 1963; Kirsh and Kissling, 1963; Netter and Lepeyre, 1964), the techniques have not been utilized for the quantitative determination of infective virus particles. The development of an assay for variola virus by the fluorescent cell-counting technique which is highly precise, sensitive, and reproducible extends the scope of the technique for the assay of additional viruses. An outstanding feature of the procedure is the ability to obtain estimates of virus infectivity in less than 24 hr. That the technique may be applicable to other poxviruses is suggested from preliminary experiments made in the course of this investigation. Fluorescent viral antigen was noted in MIcCoy cell monolayers after infection with cowpox, monkeypox, alastrim, vaccinia, or rabbitpox viruses. The similar appearance and distribution of fluorescent viral antigen in the cytoplasm of cells infected by these poxviruses precludes their differentiation from one another on the basis of immunofluorescnece. The use of centrifugal force in the fluorescent cell-counting procedure for promoting adsorption of virus inoculum onto cell monolayers, demonstrated in this study and, previously, with psittacosis virus (Hahon and Nakamura, 1964; Hahon and Cooke, 1965), offers several advantages over adsorption in a stationary position at 35 C for several hours. By the former procedure, adsorption is efficient and rapid (complete within 15 min at 25 C), the effect of thermal inactivation on infective virus is minimized, and the ability to detect virus particles from dilute suspensions may be enhanced by increasing the volume of inoculum. These features contribute to the sensitivity of the assay. Virus adsorption augmented by centrifugation also provides for the synchronous infection of cells by virus. This is desirable in studies to characterize the various developmental stages involved in the formation of mature virus particles. The incubation period is an integral part of the fluorescent cell-counting technique. In this APPL. MICROBIOL. study, it is defined as the interval between virus inoculation and the development of recognizable quantities of fluorescent viral antigen in cells. Ideally, this period should be terminated before newly synthesized virus particles are released (to prevent the secondary infection of cells) but should be sufficient to insure the formation of substantial amounts of viral antigen in primary infected cells. The concomitant increase in density of fluorescent viral antigen and the uniformity of fluorescent cell counts from 16 to 2 hr after infection established this interval as the incubation period. The time sequence of the development of viral antigen, visualized by fluorescent-antibody staining, was in agreement with the observations reported on the formation- of variola virus antigen in monkey kidney cells (Avakyan et al., 1961) and, in general, on the development of a related poxvirus, vaccinia (Noyes and Watson, 1955; Cairns, 196; Loh and Riggs, 1961). In addition to the assay of variola virus and other poxviruses, the fluorescent cell-counting technique is applicable to other facets of research with these viruses. Preliminary tests to determine the viral specificity of fluorescent cells revealed that the neutralization of variola virus infectivity by immune serum resulted in a significant reduction of fluorescent cells. A quantitative linear relationship between the reduction of fluorescent cells and dilution of immune serum has been reported previously (Hahon and Nakamura, 1964). These findings augur well for the feasibility of developing a rapid fluorescent cellcounting neutralization test to measure poxvirus serum-neutralizing antibodies. ACKNOWLEDGMENTS I gratefully acknowledge the technical assistance of Kenneth Cooke and Edmund Kozikowski during certain phases of the work. LITERATURE CITED AVAKYAN, A. A., A. D. AL'TSHTEN, F. M. KIRIL- LOVA, AND A. F. BYKOVSKII Ways of improving the laboratory diagnosis of smallpox. Prob. Virol. 6: CAIRNS, J The initiation of vaccinia infection. Virology 11: FERNANDES, M. V Irradiation of cells in tissue culture. VII. Studies on the susceptibility to bluetongue virus on radiation-induced giant cell in vitro. Z. Zellforsch. Mikroskop. Anat. 5: GOODHEART, C. R., AND L. B. JAROSS Human cytomegalovirus. Assay by counting infected cells. Virology 19: HAHON, N Cytopathogenicity and propagation of variola virus in tissue culture. J. Immunol. 81: HAHON, N., AND K.. COOKE Fluorescent Downloaded from on September 25, 218 by guest

7 VOL. 13, 1965 ASSAY OF VARIOLA VIRUS 871 cell-counting neutralization test for psittacosis. J. Bacteriol. 89: HAHON, N., AND R. M. NAKAMURA Quantitative assay of psittacosis virus by the fluorescent cell-counting technique. Virology 23: HAHON, N., M. RATNER, AND E. KOZIKOWSKI Factors influencing variola virus growth on the chorioallantoic membrane of embryonated eggs. J. Bacteriol. 75: KIRSH, D., AND R. KISSLING Use of immunofluorescence in the rapid presumptive diagnosis of variola. Bull. World Health Organ. 29: LOH, P. C., AND J. L. RIGGS Demonstration of the sequential development of vaccinial antigens and virus in infected cells: observations with cytochemical and differential fluorescent procedures. J. Exptl. Med. 114: MURRAY, H. G. S The diagnosis of smallpox by immunofluorescence. Lancet 1: NETTER, R., AND D. LAPEYRE Le diagnostic de la variole au laboratoire. Rev. Hyg. Med. Soc. 12: NoYEs, W. F., AND B. K. WATSON Studies on the increase of vaccine virus in cultured human cells by means of the fluorescent antibody technique. J. Exptl. Med. 12: PMLIPSON, L Adenovirus assay by the fluorescent cell-counting procedure. Virology 15: PIRSCH, J. B., AND E. H. PURLSON A tissue culture assay for variola virus based upon the enumeration of hyperplastic foci. J. Immunol. 89: REED, L. J., AND H. MUENCH Simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27: RIGGS, J. L., R. J. SEIWALD, J. H. BURCKHALTER, C. M. DOWNS, AND T. G. METCALF Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am. J. Pathol. 34: WHEELOCK, E. F., AND I. TAMM Enumeration of cell infecting particles of Newcastle disease virus by the fluorescent antibody technique. J. Exptl. Med. 113: Downloaded from on September 25, 218 by guest