An Electron Microscope Study of Tissue Cultures Infected with Visna.Virus

Transcription

1 VIROLOGY 14, (1961) An Electron Microscope Study of Tissue Cultures Infected with Visna.Virus HALLDOR THORMAR Institute for Expetimental Pathology, University of Iceland, Keldur, Reykjavik Accepted April 26, 1961 Sections of tissue culture cells infected with visna virus were studied in the electron microscope. The ultrastructure of the nuclei and the cytoplasm of infected cells was found to look much like that of uninfected control cells. In the former, however, a number of spherical particles was seen on the external cell surface. The particles averaged 85 rnp in diameter, were bounded by a single membrane, and contained a centrally located electron-dense core. They were found in three types of tissue cultures infected with various strains of the virus. The particles appeared 2-3 days after inoculation, concurrently with the typical cytopathic changes and the rise in virus titer of the fluid. They seemed to be formed by a budding process in the cell membrane and to be released as double-walled bodies which later developed to form the characteristic particles. It is suggested that the particles represent visna virus, and their apparent similarity to certain cancer viruses is pointed out. INTRODUCTION Visna is a virus disease of the central nervous system of sheep, causing demyelination and destruction of white matter in the brain, the cerebellum, and the spinal cord. The disease, which has been described by Sigurdsson and associates (1957) and Sigurdsson and Palsson (1958)) develops extremely slowly, clinical signs often appearing months or even years after infection, Visna virus has been propagated in tissue culture, where the characteristic cytopathic changes are found to be formation of multinuclear giant cells, often with long processes stretching from the surface (Sigurdsson et al., 1960). If tissue cultures are inoculated with large amounts of virus, almost every cell in the cultures will be typically affected Work supported by the Icelandic Science Foundation and by a grant (No. B-2425) of the Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Maryland. Present address: Statens Seruminstitut, Amager Boulevard 80, Copenhagen S., Denmark. 463 in 4-5 days. Not until a couple of days later do the cells begin to detach from the glass wall. Since a high percentage of the cells in a visna-infected tissue culture shows typical cytopathic changes before the cell sheet begins to degenerate and come off the glass, it was considered that such a culture might be suited for an electron microscope study of the ultrastructure of infected cells. The study did not, however, reveal any characteristic structural changes in the nucleus or in the cytoplasm of the cells. On the other hand, characteristic viruslike particles were seen to accumulate on the external surface of the cells in an infected culture. In the present communication, these particles will be described and data will be presented which suggest that they represent infectious visna virus. MATERIALS AND METHODS Tissue culture. Monolayer cultures of serially propagated cells derived from the choroid plexuses of sheep brain were used for most of the present work. The method

2 464 THORMAR of cultivation has been described previously night at 45. Sections were cut on a Camin detail (Sigurdsson et az., 1960). These bridge Micro Section Rocking microtome, cultures will hereinafter be referred to AS using glass knives. They were floated onto plexus cultures. the surfacr of a loc/;~ mixture of acetone in Cultures of kidney epithelial cells and of water, spread with xylene vapor (Satir and liver cells from sheep were also employed. Peachy, 1958) and picked up on Formvar- They were prepared by trypsinization of coated copper grids. Before examination in small pieces from the cortex of the kidney the electron microscope, which \vas a Sicand from the liver. All cultures were grown mens Elmiscope I, the sections were usually in roller tubes using medium 199 with 20% stained with a saturated solution of lead sheep serum as growth medium. The main- acetate, according to the method of Watson tenance medium in the inoculated cultures (1958). consisted of medium 199 with 2% sheep serum. All media contained 100 units of RESULTS penicillin and 100 pg of strept,omycin per UT&infected Plexus Cultures milliliter. Virus. Strain K485 of visna virus adapted Plexus cultures incubated with mainteto growth in tissue culture (TC) through nance medium at 37 for 6-7 days served as more than 30 passages (Sigurdsson et al., control cultures to be compared with cul- 1960) was used in the greater part of the tures infected with visna virus. At the time present work. Strains K643, 644, 682, and of fixation for electron microscopy, the un- 796 in their eleventh or twelfth TC passage infected cultures appeared normal, as obwere used in a few cases. served in the light microscope. The virus was titrated by inoculating ten- In the electron microscope, the cytoplasm fold dilutions of infectious TC fluid into of uninfected cells varied considerably in roller tube cultures (Thormar, 1961) ; the appearance from one culture to another, but 50% infectivity end point was calculated by usually looked rather poor in structural the method of Reed and Muench. Infectious constituents. Only a few mitochondria were TC fluid was employed for inoculation of seen. Ergastoplasm was more often found cell cultures used for electron microscopy. well preserved, and the cytoplasm often The fluid had an infectivity titer of IO6 to contained a number of small vacuoles. The lo7 TCID60 per 0.1 ml, and the inocula were nuclei showed a finely dispersed matrix, and chosen so that they would contain more nucleoli were sometimes observed. than one infectious unit per cell. Preparation of specimens for electron mi- Plexus Cultures Infected with Visna Virus croscopy. Routine methods were used for Plexus cultures inoculated with infectious preparation of tissue cultures for electron TC fluid showed widespread cytopathic microscopy. The cells were rinsed once in changes, by light microscopy, after incuba- Hanks salt solution and fixed at 4 for 15- tion at 37 for 6-7 days (Fig. 1). They were 20 minutes, using 1% solution of osmium then fixed and prepared for electron microstetroxide, buffered at ph , according copy. to the method of Palade. After fixation, the In the electron microscope the cytoplasm cells were rinsed quickly with distilled wa- of cells from infected cultures looked simter and covered with 70% ethanol, The cell ilar to that of uninfected cells, appearing layer was then detached from the glass wall rather lacking in electron-dense structure. with a metal spatula. It came off as small Mitochondria were sometimes seen but were flakes that were transferred to a centrifuge usually not well preserved, showing swelltube and collected by low speed centrifuga- ing and vacuolization. Ergastoplasm was obtion, The cells were dehydrated in graded served in most of the cells. The internal dilutions of ethanol and embedded in a mix- structure of the nuclei was similar to the ture of 75% butyl and 25% methyl meth- structure of nuclei of uninfected cells. acrylate, containing 2% benzoyl peroxide. The most striking difference between un- The polymerization was carried out ovcr- infected and infected cultures, as revealed

3 VISNA VIRUS INFECTION 465 FIG. 1. Plexus culture 6 days after inoculation with visna virus. The characteristic cytopathic changes consist in formation of multinucleated cell syncytia, the nuclei being arranged in a rosette-like pattern. Most of the cells in this culture show pathologic effects. Giemsa-stained. Magnification : X 146. by electron microscopy, was the appearance in the latter of small round particles on the external surface of the cells. Often the particles lay side by side along the cell membranes and sometimes they formed large clusters, containing dozens of particles. Figure 2 shows a section through cells from an infected plexus culture. A few round particles, each with a dense central body, can be seen scattered along the surface of the cells, and at the top of the figure there is a cluster of particles. A swollen and disrupted mitochondrion is seen at the lower right corner, and remnants of ergastoplasm and mitochondria are found scattered through the vacuolized cytoplasm. Occurrence of Extracellular Particles in Infected Kidney and Liver Cell Cultures Confluent monolayers of kidney cells were inoculated with visna virus and incubated at 37. After 6-7 days, cytopathic changes with the formation of multinuclear giant cells were seen by light microscopy of fixed and Giemsa-stained infected cultures. The cytopathic changes increased during the following days and the infectivity titer of the culture fluids rose to lo6 to 10 TCIDsO per 0.1 ml. On the seventh to tenth day of infection, kidney cell cultures were prepared for electron microscopy together with normal-looking uninfected cultures of the same age. Electron microscopic examination of

4 THORMAR FIG. 2. Section from a plexus culture fixed 7 days after inoculntion Visna particles can be seen on the external cell surface. Magnific.ation: sections from the infected cultures showed extracellular particles having the same appearance as those observed in infected plexus cultures. No particles of this type were seen in sections of uninfected kidney cell cultures, which resembled sections from infected cultures with respect to the ultrastructure of the cytoplasm and the nuclei. Monolayer cultures of liver cells inoculated with visna virus showed cytopathic X with \-isna virus. 22,000. changes similar to those seen by light microscopy of infected plexus and kidney cell cultures. Electron microscopic examination of the infected liver cell cultures revealed the presence of round extracellular particles, containing a dark central body. Altogether, more than twenty plexus, kidney, and liver cell cultures infected with visna virus have been examined in the electron microscope. In all these cultures the

5 characteristic particles were seen, usually in great number. They have never been observed in uninfected cultures, although these have been thoroughly searched. These particles will, in the following sections, be referred to as visna particles. The Size and the Structure Particles VISNA VIRUS INFECTION 467 of the Visna Figure 3 shows a cluster of visna particles tt a magnification of 120,000 times. The Iarticles seem to have a slightly ovoid shape, but as the longest axes of most of ;hem are oriented in the same direction, it is relieved that the apparent ovoid shape is lue to compression of the section, caused by (he microtome knife, and that the particles Ire in fact spherical. The irregular shape of :ome of the particles might be caused by meven polymerization or shrinkage of the nethacrylate. The size of the visna particles was estinated by measuring their diameters in mirographs. About 200 particles were meas- (red and in each one the average between he longest and the shortest axis was conidered to be the true diameter. The average liameter of all particles measured was 85 np. About 90% were within the range of O-100 rnp and the remaining 10% were ither slightly smaller or bigger. Particles onsiderably bigger than 100 rnp in diameter Jere seen occasionally. The visna particles are bounded by an pparently single membrane and contain a entrally located osmiophilic core, separated rom the outer membrane by a zone of low lectron density. The central cores seem also 3 be surrounded by a membrane. Their verage diameter was found to be 35 rnp nd about 80% of those measured ranged in ize from 30 to 40 rnp. No difference was found in the appearrice of particles seen in the various types f tissue culture, either in size or structure. ormation of Spherical Bodies at the Surface of the Cells enveloped by double membranes are sometimes found intermingled with visna particles near the surface of the cells (Fig. 5). Figures 6 and 7 show a number of protrusions of the cell membrane, varying in size. Some appear as a slight thickening and bulging of t he membrane, others have protruded farther, having the shape of hemispherical buds. Still others are assuming a spherical shape or seem to be breaking away from the membrane as double-walled spherical bodies. The various protrusions apparently represent steps in t,he formation of the free extracellular bodies, which are seen near the surface of the cells together with visna particles. The phenomena seen in Figs. 6 and 7 are illustrated at a higher magnification in Figs. 8 and 9. In Fig. 8 a small bulge is seen at the right border of the field, two buds are seen near the center, and at the left a spherical body is seemingly detaching from the membrane. A dark dot is visible in the center of this body and in the one shown in the inset. Figure 9 shows a couple of buds of almost hemispherical shape and a bud assuming a spherical shape and separating from the cell surface. Above it two visna particles are found. Free spherical bodies are visible near the right border of the field and at the lower left corner. Figures 8 and 9 show that the inner membrane of the double-walled buds and bodies is more electron dense than the outer. The extracellular bodies have the same shape as visna particles but appear on the whole slightly bigger. Some of them are seen to have a structure similar to that of visna particles, containing a dense central core, which is, however, much smaller and less distinct than the central core of visna particles (cf. Fig. 8). The bodies appear in sections of infected cultures more rarely than do visna particles and always accompanied by the latter. The buds and the extracellular bodies are believed to represent precursors of the visna particles. 1 Relationship between Xumber of Visna Particles and Infectious Virus Titer In sections of tissue cultures infected with isna virus, cell membranes are often seen 1 be covered with a great number of ouble-walled buds (Fig. 41. Also, in in- In order to study if there were a relation- :cted cultures, small extracellular bodies ship between the number of visna particles

6 THORMAR 468 FIG. 3. Cluster of visna Magnification : X 120,000. particles from a plexus culture fixed 7 days after inoculation

7 VISNA FIG. 4. Cell double-walled FIG. 5. The walled bodies which apparently VIRUS 469 INFECTION from a plexus culture 7 days after inoculation. The membrane buds. Magnification : X 22,000. same culture as in Fig. 4. Visna particles are seen intermingled near the external cell surface. A few buds are also seen on the is disintegrating. Magnification: X 22,000. is covered cell with with dou Iblemembr ane,

8 470 THORMAR FIG. 6. Cell from a plexus culture 7 days after inoculation, showing protrusions of the, cell membrane which are believed to represent stages in the formation of free extracelluh11 bodies. A few visna particles are also seen near the cell surface. Magnification: x 22,000. FIG. 7. Plexus cultures 7 days after inoculation, showing the same phenomenon as in Fig. 6. Buds of various lengths are protruding from the cell membrane, some of them pinching off to form spherical bodies. A great number of visna particles are seen covering the cell surface. The section seems to be a little compressed. Magnification: X 22,000.

9 VISNA VIRUS INFECTION FIG. 8. Section of a plexus culture 7 days after inoculation, showing a piece of cell membrane. Protrusions in various stages of their development into free bodies are seen along the membrane, and in the inset an apparently free spherical body from the same section is shown. Note the dense centers of the bodies. Magnification: X 120,000. seen in an infected tissue culture and the virus titer of the surrounding fluid the following experiment was made: A number of young monolayer plexus cultures were inoculated, each with 0.1 ml of infectious TC fluid, and incubated in a roller drum at 37 for 6 hours, a period which has been found sufficient to ensure maximum adsorption of virus (unpublished data). One culture was then fixed for electron microscopy. The remaining cultures were washed with six changes of medium 199 and the fluid of the sixth washing was saved for titration of virus. The cultures were then incubated further and at daily intervals for 7 days one culture was harvested for fixation. The fluid from each tube was saved, deep-frozen, and later assayed for infectious virus. About half of the cell layer in each culture was detached from the glass and prepared for electron microscopy, while the remaining cells were Giemsa-stained for examination by light microscopy. Sections of about 100 different cells from each culture were examined in the electron microscope, and all visna particles seen in 3-4 sections of each of the 100 cells were counted. The results are summarized in Table 1 and Fig. 10. Table 1 shows that visna particles appear in a detectable number on the second to the third day of infection concurrently with the development of cytopathic changes in the culture and at the same time as the main rise in virus titer occurs in the fluid, i.e., from 1000 to l,ooo,ooo TCID5,, per 0.1 ml. As illustrated in Fig. 10, the number of particles increases from the second to the fifth day of infection, almost in parallel with the infectious virus titer. After this time, the titer did not rise sig-

10 THORMAR FIG. 9. Another section from the same culture as in Fig. 8, showing cell membrane with buds, free bodies, and two visna particles. The buds and the bodies arc bounded by double membranes, the inner membrane being more distinct than the outer. Magnification: :a nificantly but the number of visna particles and the cytopathic changes increased until the end of the experiment. Four days after inoculation, visna particles were seen on the surface of almost every cell examined in the electron microscope, but only one or very few in each section. On the fifth to the seventh day the number of particles surrounding a cell was greatly increased, reaching a maximum on the seventh day, when clusters of particles were often seen. On the sixth and the seventh day, cell membranes were often found to be covered with the small buds that are believed to represent precursors of visna particles being released from the cells. Visna particles were not observed within the cells at any time, either in the cytoplasm or in the nucleus. This experiment has been repeated, both with plexus cultures and with cultures of kidney cells, always with similar results. DISCUSSION The present study has shown that infection of tissue cultures with visna virus causes the appearance of characteristic particles which accumulate on the surface of the cells. The particles have been observed by electron microscopy of plexus cultures infected with five strains of visna virus which have been propagated in TC, and they have been seen in cultures of sheep kidney and liver cells inoculated with visna virus. These three types of cell cultures support the growth of the virus and undergo similar cytopathic changes. The size and the structure of the particles is the same regardless of the strain of visna virus used for infection and of the t,ype of tissue culture employed.

11 VISNA VIRUS INFECTION 473 TABLE 1 CYTOPATHIC CHANG s, RELATIVE NUMBER OF VISNA PARTICLEG, AND VIRUS TITER IN PLEXUS CULTURES HARVESTED AT VARIOUS TIMES AFTER INOCULATION WITH VISNA VIRUS= Time of harvest Cytopathic (CP) changes Relative number of visna particles Virus titer of fluid 6 Hours None No particles observed 1O.Ob 30 Hours None No particles observed 103JJ 2 Days Slight CP changes; 5-10% of the cells with two or more nuclei (2) Days Increased CP changes; 4O-5Oyo of the cells multinuclear or of stellate form o 4 Days 5 Days 6 Days 7 Days 50-60~0 of the cells with CP changes 70-8Oojo of the cells with CP changes Cells beginning to degenerate and detach from the glass wall Increasing disintegration, but multinuclear and giant cells still present in great number J r Further increase 106. Innumerable 10.o (1 Cytopathic changes were studied by light microscopy of Giemsa-stained cultures. Relative number of visna particles in the various cultures was obtained by counting all the particles seen in sections from about 100 cells from each culture, using 34 sections of each cell. The infectious virus titer is expressed as TCIDw per 0.1 ml of culture fluid. - b Fluid of the sixth washing. The visna particles are viruslike, being spherical and containing a dense internal body. Their size is within the size range of viruses, the average diameter being about 85 rnp. There is a considerable variation between individual particles but definite size groups have not been found. The relative size of the internal bodies varies somewhat, but as a rule their diameter is less than one half of the diameter of the particles. In the present study, visna particles were seen in all tissue cultures infected with visna virus and showing widespread cytopathic changes. They have, on the other hand, not been found in uninfected control cultures. This fact, together with their viruslike structure, suggests that the particles represent visna virus. The observations (1) that visna particles do not appear in a detectable number in infected tissue cultures until the cells have begun to release virus, and (2) that the number of visna particles increases during the infection in a similar manner as the infectious virus titer render a further indication in favor of the viral nature of the particles. The apparent similarity between visna particles and the double-walled spherical bodies formed by budding of the cell membrane, and their appearance together on the surface of the cells, suggests a relationship between the bodies and visna particles, possibly that the former are precursors of the latter. This is in agreement with the fact that the particles have never been observed intracellularly, although found in a great number on the external cell surface. Structures likely to be precursors of visna particles have not been observed within the cells either. However, on the basis of the present work no conclusion can be drawn concerning the effect of visna infection on the ultrastructure of tissue culture cells. The cytoplasm of infected and control cells had

12 474 THORMAR FIG. 10. Increase in virus titer (left ordinate) and in relative number of visna particles (right., ordinate) in tissue cultures incubated at 37 for various time periods after inoculation with visna virus (abscissa). a similar appearance, as observed by electron microscopy, looking rather poor in electron dense structure. Whether this lack of inner structure is due to an unspecific degeneration of the tissue culture cells, to a disruption of the cell components during the polymerization of methacrylate, or to some other cause is not known at the present time. Considering further the suggestion that visna particles are formed by a budding process in the cell membrane and are released as double-walled spherical bodies, it should be noted that intermittent stages between these and mature visna particles have not been observed in the electron microscope. However, double-walled bodies with a dense center surrounded by more diffuse material have been seen (cf. Fig. 8). Since the bodies often appear somewhat bigger than most of the visna particles, a contraction of their double membrane might be the last step in the process of particle formation, occurring so rapidly that there would be little probability of fixing a body during this step. If the inner membrane contracts more than the outer, a dense central core will be formed, leaving a zone of lesser density between the core and the outer membrane. Assuming that the visna particles represent visna virus, the present electron microscope study thus suggests that visna virus is formed at the surface of the host cells at the time of extrusion. Franklin (1958) has put forward the hypothesis that animal viruses which contain lipids as an essential component are completed as they pass to the external surface of the host cell by obtaining a lipid coat from the cell membrane. Such viruseswhich include, for example, myxoviruses, Western and Eastern equine encephalomyelitis viruses, and certain cancer virusesare released continuously from their host cells over a long period of time unlike those viruses which are released by a burst (Franklin, 1958). Visna virus seems to fit into this pattern, since it has been found to be ether sensitive (Thormar, 1961), thus indicating that it is lipid containing, and the viral particles are apparently formed at the cell surface, as suggested by the present work. Furthermore, the observation that tissue culture cells show cytopathic changes,

13 VISNA VIRUS INFECTION 475 by light microscopy, shortly after inoculation with visna virus and remain intact on the glass wall for at least 5-6 days of incubation while the virus titer in the fluid gradually rises to maximum (cf. Table 1) is compatible with the conception of continuous virus release over a long period of time. The protrusions observed on the surface of cells infected with visna virus show a striking resemblance to those seen on the surface of mouse mammary carcinoma cells (Lasfargues et al., 1959; Moore et al., 1959; Goldfeder et al., 1960)) and mouse leukemic cells (de Harven and Friend, 1960). In the case of these tumor cells, the protrusions also develop into double-walled bodies which are released from the cell membrane. In some instances, these bodies-which are believed to represent viral particles-later seem to be rearranged to form a sac, containing a central nucleoid (Lasfargues et al., 1959; Goldfeder et al., 1960). In this connection it is of interest to note that visna infection in sheep seems to have certain characteristics in common with mouse mammary carcinoma and mouse leukemia, namely a long initial period of latency and-once clinical signs have appeared-a slow progressive course which leads to death. These are the characteristics of slow infections, as defined by Sigurdsson (1954). Furthermore, it is noteworthy for visna infections that they have in many instances been found not to cause formation of circulating neutralizing antibodies (Sigurdsson et az., 1960), apparently also a characteristic of the above-mentioned neoplastic diseases (Furth and Metcalf, 1958). Finally, as suggested by the present work, visna virus grown in tissue culture seems to be formed by a budding process similar to that reported in the case of the mouse mammary tumor agent and of Friend leukemia virus, the viral membrane being derived from the membrane of the host cell. It would be interesting if this peculiar mechanism of virus formation and a low antigenicity of the virus turned out to be characteristic traits of slow virus infections, in addition to the protracted course of the disease. ACKNOWLEDGMENTS The author wants to thank Dr. Halldor Grimsson for much help during the work with the electron microscope. I arn grateful to Dr. Herdis von Magnus and civil engineer Aksel Birch-Andersen, Statens Seruminstitut, Copenhagen, Denmark, for valuable criticism of the manuscript, and to Miss Anne-Grete Overgaard for preparation of the photographic prints. REFERENCES DE HARVEN, E., and FRIEND, C. (1960). Further electron microscope studies of a mouse leukemia induced by cell-free filtrates. J. Biophys. Biothem. Cytol. 7, FRANKLIN, R. M. (1958). An hypothesis to explain the relation between the synthesis and release of animal viruses from infected cells and the lipid content of the viruses. Experientia 14, FURTH, J., and METCALF, D. (1958). An appraisal of tumor-virus problems. J. Chronic Diseases, 8, GOLDFEDER, A., GELBER, D., and MOORE, D. H. (1960). An electron microscope study of spontaneous mammary carcinomas in a subline of strain DBB mice. J. Natl. Cancer Inst. 25, LASFARGUES, E. Y., MOORE, D. H., MURRAY, M. R., HAAGENSEN, C. D., and POLLARD, E. C. (1959). Production of the milk agent in cultures of mammary carcinoma. J. Biophys. Biochem. Cytol. 5, MOORE, D. H., LASFARGUES, E. Y., MURRAY, M. R., HAAGENSEN, C. D., and POLLARD, E. C. (1959). Correlation of physical and biological properties of mouse mammary tumor agent. J. Biophys. Biochem. Cytol. 5, SATIR, P. G., and PEACHEY, D. D. (1958). Thin sections. II. A simple method for reducing compression artifacts. J. Biophys. Biochem. Cytol. 4, SIGURDSSON, B. (1954). Rida, a chronic encephalitis of sheep. With general remarks on infections which develop slowly and some of their special characteristics. Brit. Vet. J. 110, SIGURDSSON, B., and P~LSSON, P. A. (1958). Visna of sheep. A slow, demyelinating infection. Btit. J. Exptl. Pathol. 39, SIGURDSSON, B., P~~LSSON, P. A., and GR~MSSON, H. (1957). Visna, a demyelinating transmissible disease of sheep. J. Neuropathol. Exptl. Neurol. 16, SIGURDSSON, B., THORMAR, H., and P&,soN, P. A. (1960). Cultivation of visna virus in tissue culture. Arch. ges. Virusforsch. 10, THORMAR, H. (1961). Stability of visna virus in infectious tissue culture fluid. Arch. ges. Virusforsch. 10,501~509. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. II. Application of solutions containing lead and barium. J. Biophys. Biochem. Cytol. 4,