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Electron microscopical aspects of hemadsorption in tissue cultures infected with influenza virus
VIROLOGY
6, 689-701 (19%)
Electron Microscopical Aspects of Hemadsorption in Tissue Cultures Infected with Influenza Virus JOHN E. HOTCHIN,SOPHIA M. COHEX,HELMUT A4ND CARL.4 RUSKA' Division
of Laboratories
and Research, hTew York State Department
RUSKA,* of Hea,lth, Albany
Accepted -4ugust 1, 1958
Adsorption of guinea pig ergt.hrocgtes to monkey kidney cells in tissue culture infected with influenza virus was found by electron microscopy to occur by two processes. In one, the red cells were attached to virus filaments protruding from the host cell cytoplasmic membrane, and the red cell and host cell were separated by a distance of up to Ip. This type of attachment is similar to that seen in hemagglutination. In the other, there was direct and close binding of the eryt,hrocytes to the infected monkey kidney cell in the absence of detectable virus filaments or particles. The significance of the finding of an apparent. change in the immunologically specific structure of the surface of the host cell following infection by influenza virus is discussed. INTRODUCTTOX During an investigation on the maturation and release of influenza virus from monkey kidney cells, use was made of the hemadsorpt,ion (Vogel and Shelokov, 1957) t’o locate cells which showed evitechnique dence of infection following exposure to virus. When erythrocytes are
added directly to monolayer cell cultures infected with influenza virus, the red cells are adsorbed to the host cells. This hemadsorption can easily be seen in t’he light microscope; it occurs before cytopathic changes and is specifically inhibited by antiviral serum; it occurs only upon cells infected with the virus, and not upon normal control rells. Areas of cultures shelving hemadsorption were sectioned and examined by electron microscopy. MATERIALS
ASD METHODS
Tissue Culture Stationary monolayer cultures prepared from trypsinized monkey kidney were used (Youngner, 1954). The medium for cell growth, 1 Present address: Jnstitut fiir Elektronenmikroskopie, 689
Diisseldorf,
Germany.
690
HOTCHIN,
COHEN,
RUSKA
.4ND
RUSKA
similar to Bodian’s (1956), contained 2 % calf serum. The cultures were washed at least twice before inoculation. Several tissue culture t’ubes from each lot were tested for hemadsorption with guinea pig erythrocytes to avoid the use of cultures containing simian hemagglutinating agents. Such an agent was recovered from one lot. For the tests with influenza virus, the medium was Eagle’s (1955; Eagle et al., 1956) with 1% glucose, 0.1% yeast, extract, and antibiotics, The cultures were incubated at 35”.
Influenza virus type A Asian, strain No. 5794 (A/Albany 4/57), was isolated in this laboratory. Inoculum was infected allantoic fluid from the third passage in embryonated hens’ eggs subsequent to one passage in monkey kidney tissue culture. Hemadsorption The methods of Vogel and Shelokov (1957) were followed except that after removal of the nutrient fluid from a tissue culture tube, the cell sheet was washed with Hanks’ balanced salt solution or nutrient fluid
I
FIXATION
e>
POLYMERIZATION
CELL SHEET
I MOUNTING
OF SPECIMEN
SPECIMEN TO BE CUT FIG.
1. Preparation
and mounting of specimens.
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
691
before the addition of 0.2 ml of a 0.4 % suspension of guinea pig erythrocytes in 0.85 % NeCl solution. Readings were made after 10 minutes at room temperature. After 30 minutes, the tubes were washed to remove unadsorhed red cells, fresh fluid was added, and the reaction read again.
FIG. 2. Cyto-hemadsorption. Contact between the surfaces of red blood cells and kidney culture cell (m = mitochondria, N = nucleus, IJ = vesicles). Magnification: X 10,000.
692
HOTCHIN,
COHEN,
The final washed preparation studies.
RUSKA
AND
RUSKA
was used for the electron
microscope
Electron Microscopy The fluid was removed and the tubes were placed horizontally with the cell sheet on the uppermost side of the tube. Approximately 1 ml of aqueous 2% osmium tetroxide solution was introduced into the tube beneath the cells (Fig. l), which were fixed in the vapor for 10 to 15 minutes. Residual osmium tetroxide solution was washed three times from the glass wall with isotonic buffer. The fixed cells were dehydrated with graduated alcohols in 15 minutes and covered with butyl methacrylate for W hour (three changes). They were then immersed in butyl methacrylate with 2 % Lucid01 for 20 minut,es and embedded in pre-
FIG.
3. Cyto-hemadsorption.
Magnification:
X 16,250.
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
6%
FIG. 4. Cyto-hemadsorption and virus-hemadsorption. Upper red cell is adsorbed by contact of cell membranes. At lower right, red cell is attached to culture cell by interaction of virus filaments in stats nascendi (arrows). Magnification: X 22,500.
polymerized butyl methacrylate. Before final polymerization under nitrogen, the tubes were placed almost horizontally so that the cell sheet was covered with a wedge-shaped layer of embedding medium with a maximum thickness of 5 mm. To remove the plastic from the glass tube, t’he closed t’ubes were placed for a few minutes in a dry-ice-alcohol mixture. This causes t,he plastic to separate from the glass wall and the tube breaks with a loud crack. The area of plastic cont’aining t,he cells
694
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
FIG. 5. Cyto-hemadsorption, virus-hemadsorption, and hemagglutination. Border zones of two tissue culture cells. The upper cell shows cyto- and virus-hemadsorption. Along the lower cell virus-hemadsorption and hemagglutination are mediated by virus filaments. Magnification: X 10,000.
was cut into slices of l-3 mm thickness and the slices were cemented on top of a conventional plastic block to hold the specimen in the microtome. The surface to be cut was reduced to $$ mm >( l-l% mm (Fig. 1). A Porter-Blum microtome with a diamond knife (obtained from the Venezuelan Institute for Scientific Investigation, Caracas) was used for sectioning, and a Siemens Elmiskop I for the electron microscopy. Figures 2-8 were made of a tissue culture inoculated with about lo6
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
695
50 % egg infective doses of the influenza virus and tested for hemadsorption after 24 hours’ incubation. Figure 9 was from a culture incubated for 6 hours after infection. RESULTS
Electron microscopy revealed that red cells were adsorbed to virusinfected monkey kidney cells by t,wo different processes. In one case guinea pig red cells were frequently tightly bound to the surface of
FIG. 6. Virlls-hemadsorption and hemagglutination culture cell. hlaguification: X 10,000.
along
surface
of kidney
696
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
FIG. 7. Virus particles adsorbed on the red cells near surface of kidney culture cell. Red cells are slightly compressed. Note the numerous filamentous, tubular forms of virus and their close proximity along the surface of red cells. Magnification: X 11,250.
infected tissue culture cells in areas where no filamentous processes were seen (Figs. 2 and 3). The red cells were flattened onto the influenza virushost cell and even appeared in some areas to be drawn into the host cell or to have caused a depression or protrusion of the cell surface. Such attachment was direct between the red cell and monkey kidney cell and involved the cell membrane only, wit,h no intermediary virus. In the second type of attachment, red cells were adsorbed to the virus filaments
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZS
VIRUS
697
which protruded from the infected monkey kidney cells. Both types of at’tachment could be seen on some cells (Figs. 4 and 5). In some sections hemagglutination could be seen as red cells with virus bridges between them; here, the red cells were separated by a distance of up to 1 P. A similar dist’ance separated red cells from host cells when t’hey were attached by virus filaments (Figs. 5-7). Control sections of red cells agglutinated by the same strain of influenza virus in allantoic fluid also showed virus bridges between the cells.
FIG. 8. Beginning of virus release and virus-hemadsorption. Note that cytoplasmic and nuclear structures of the host cell are better preserved than in cells which show cyto-hemadsorption. Magnification: X 11,250.
698
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
FIG. 9. Kidney culture cell of almost normal appearance 6 hours after in. fection of the culture. Note the dense mitochondria (m) and the parallel membranes of the endoplasmic reticulum (er). A Golgi zone (G) is visible in the center of the cytoplasm. Magnification: X 11,250.
The two forms of hemadsorption described differ from each other in respect to the presence or absence of an intermediary agent-in this case virus. Both forms differ from hemagglutination in which only one cell type is involved (Fig. 10). We have used the term hemagglutination in the usual sense, restricted to the binding together of red cells by virus. For hemadsorption of the direct type involving red cells attached intimately to the host cell membrane without visible virus we propose the
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
6’39
term “cyto-hemadsorption.” The indirect type, where virus filaments protruding as part of the host cell are attached to a red cell, we have These two forms of attachment can be called “virus-hemadsorption.” distinguished only by electron microscopy. The structure of host cells which showed cyto-hemadsorption was always altered. The cytoplasm contained only a few small mitoc+hondria with a matrix of low density. Vesicles of varying size (50 rnp to several P) were seen and small granules att’ached to the membranes of the vesicles 01 scattered in the rytoplasmic matrix. The endoplasmic reticulum in its normal form with close spaces between parallel membranes had disappeared (compare lcigs. 2-8 with Fig. 9). The nuclear double membrane was preserved but, frequently interrupted, and the inner structure of t,he nuclei was abnormally loose (Figs. 2, 3, and 5). The structure and size of cells several hours after infection indicated considerable swelling (Fig. 9). Culture cells which showed virus-hemadsorption along their surface wit)h additional hemagglutination were in the state of virus release; cytoplasmic and nuclear structures in these had undergone similar but less marked changes than those seen in cells with cyto-hemadsorption (Fig. 8).
HEMAGGLUTINATION
VIRUS-HEMADSORPTION FIG. 10. Diagram
of different
forms
of hemadsorption.
700
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
The extracellular virus particles appeared either as rings with a diameter of approximately 70 rnF or as tubes of the same diameter and a length up to 2 P. In many instances virus particles were crowded upon the red cell surface so tightly as to indicate limitation only for geometrical reasons (Fig. 7). Thus, it appears that a single red cell could adsorb a very large number of spherical virus particles, possibly in the order of 10,000. DISCUSSION
Hemadsorption reactions with influenza virus in tissue culture have been found to be highly sensitive and specific in accordance with the observations of Shelokov et al. (1958). The electron micrographs suggest that after infection with influenza virus the host cell surface undergoes a change in its immunologically specific structure, rendering the surface capable of forming an attachment to red cell membranes analogous to that shown by mature infectious virus particles. This adsorption occurs only at the surface of virus-infected cells, and is absent in uninoculated controls. The effect is observed where few or no virus filaments of the type described by Morgan et al. (1956) and Bang and Isaacs (1957) are seen at the cell surface. In this respect the host cell behaves as if it were a single large virus particle. Such a change in the structure of the surface of the host cell, in which it becomes antigenically similar to the structure of the surface of the virus, may explain how the virus gains its surface immunological specificity during the last step of its formation as an extrusion of material into a tube of protruding cell membrane. This hypothesis is consistent with reported findings (Knight, 1946; Smith et al., 1955) that influenza virus contains antigens belonging to the host cell in addition to those specifically viral. The virus particle may be in fact enclosed by a membrane, which is essentially the same as that of the altered infected cell. It is possible that after infecting the cell the virus behaves as a cytoplasmic gene, the presence of which is reflected as a phenotypic change in the host which can be detected by the ability of the host cell membrane to behave in a manner similar to that of the virus membrane. The concept that the myxoviruses induce a phenotypic change in the surface of the infected host cell is being studied further. The electron microscopical findings also suggest that all parts of the surface of the red cell are capable of specific attachment to the infected host cell. Furthermore, virus particles have been seen crowded upon the surface of red cells. These observations indicate that the number of
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
701
available sites of specific attachment for virus particles may be very great, in sharp contrast to the estimate of about 300 made by Sagik et al. (1954) ; more recently this figure has been considered by Tyrrell and Valentine (1957) to be an underestimation. REFERENCES BANG, F. B., and ISAACS, A. (1957). Morphological
aspects of virus cell relationmumps and Newcastle (Mysouirt~s). In Ciba Foundation Symposium on the Nature of Viruses (G. E. W. Wolstenholme and E. C. P. Millar, eds.), pp. 249-262. Little, Brown and Company, Boston. BODIAN, I). (1956). Simplified method of dispersion of monkey kidney cells with trypsin. Virology 2, 575-576. EAGLE, H. (1955). Utilization of dipeptides by mammalian cells in tissue cult.ure. ships in influenza,
Proc. Sot. Exptl. Biol. Med. 89, 96-99. EAGLE, H., OYAMA, V. I., LEVY, M., and FREEMAN, A. (1956). Myo-inositol
as an essential growth factor for normal and malignant human cells in tissue culture. Science X%3,845-847. KNIGHT, C. A. (1946). Precipitin reactions of highly purified influenza viruses and related materials. J. Exptl. Med. 83, 281-294. MORGAN, C., ROSE, H. M., and MOORE, D. H. (1956). Structure and development of viruses observed in the electron microscope. III. Influenza virus. J. Erptl. ‘2l’ed. 104, 171-182. SAGIK, B.. PUCK, T., and LEVINE, S. (1954). Quantitative aspects of the spontaneous elution of influenza virus from red cells. J. Exptl. Med. 99, 251-260. SHELOKOV, A., VOGEL, J. E., and CHI, L. (1958). Hemadsorption (adsorpt,ionhemagglutination) test for viral agent,s in tissue culture with special reference to influenza. Proc. Sot. Exptl. Biol. Med. 97, 802-809. SMITII, W., BELYAVIN, G., and SHEFFIELD, F. W. (1955). The host-tissue component of influenza viruses. Proc. Roy. Sot. B143, 504-522. TYRRELL, I). A. J., and VALENTINE, R. C. (1957). The assay of influenza virus part,icles by haemagglutination and electron microscopy. J. Gen. Microbial. 16, 668-675. VOGEL, J., and SHELOKOV, A. (1957). Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126, 358-359. YOUNCNER, J. S. (1954). Monolayer tissue cultures. I. Preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells. hoc. Sot. Exptl.
Biol.
Med. 85, 202-205.
6, 689-701 (19%)
Electron Microscopical Aspects of Hemadsorption in Tissue Cultures Infected with Influenza Virus JOHN E. HOTCHIN,SOPHIA M. COHEX,HELMUT A4ND CARL.4 RUSKA' Division
of Laboratories
and Research, hTew York State Department
RUSKA,* of Hea,lth, Albany
Accepted -4ugust 1, 1958
Adsorption of guinea pig ergt.hrocgtes to monkey kidney cells in tissue culture infected with influenza virus was found by electron microscopy to occur by two processes. In one, the red cells were attached to virus filaments protruding from the host cell cytoplasmic membrane, and the red cell and host cell were separated by a distance of up to Ip. This type of attachment is similar to that seen in hemagglutination. In the other, there was direct and close binding of the eryt,hrocytes to the infected monkey kidney cell in the absence of detectable virus filaments or particles. The significance of the finding of an apparent. change in the immunologically specific structure of the surface of the host cell following infection by influenza virus is discussed. INTRODUCTTOX During an investigation on the maturation and release of influenza virus from monkey kidney cells, use was made of the hemadsorpt,ion (Vogel and Shelokov, 1957) t’o locate cells which showed evitechnique dence of infection following exposure to virus. When erythrocytes are
added directly to monolayer cell cultures infected with influenza virus, the red cells are adsorbed to the host cells. This hemadsorption can easily be seen in t’he light microscope; it occurs before cytopathic changes and is specifically inhibited by antiviral serum; it occurs only upon cells infected with the virus, and not upon normal control rells. Areas of cultures shelving hemadsorption were sectioned and examined by electron microscopy. MATERIALS
ASD METHODS
Tissue Culture Stationary monolayer cultures prepared from trypsinized monkey kidney were used (Youngner, 1954). The medium for cell growth, 1 Present address: Jnstitut fiir Elektronenmikroskopie, 689
Diisseldorf,
Germany.
690
HOTCHIN,
COHEN,
RUSKA
.4ND
RUSKA
similar to Bodian’s (1956), contained 2 % calf serum. The cultures were washed at least twice before inoculation. Several tissue culture t’ubes from each lot were tested for hemadsorption with guinea pig erythrocytes to avoid the use of cultures containing simian hemagglutinating agents. Such an agent was recovered from one lot. For the tests with influenza virus, the medium was Eagle’s (1955; Eagle et al., 1956) with 1% glucose, 0.1% yeast, extract, and antibiotics, The cultures were incubated at 35”.
Influenza virus type A Asian, strain No. 5794 (A/Albany 4/57), was isolated in this laboratory. Inoculum was infected allantoic fluid from the third passage in embryonated hens’ eggs subsequent to one passage in monkey kidney tissue culture. Hemadsorption The methods of Vogel and Shelokov (1957) were followed except that after removal of the nutrient fluid from a tissue culture tube, the cell sheet was washed with Hanks’ balanced salt solution or nutrient fluid
I
FIXATION
e>
POLYMERIZATION
CELL SHEET
I MOUNTING
OF SPECIMEN
SPECIMEN TO BE CUT FIG.
1. Preparation
and mounting of specimens.
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
691
before the addition of 0.2 ml of a 0.4 % suspension of guinea pig erythrocytes in 0.85 % NeCl solution. Readings were made after 10 minutes at room temperature. After 30 minutes, the tubes were washed to remove unadsorhed red cells, fresh fluid was added, and the reaction read again.
FIG. 2. Cyto-hemadsorption. Contact between the surfaces of red blood cells and kidney culture cell (m = mitochondria, N = nucleus, IJ = vesicles). Magnification: X 10,000.
692
HOTCHIN,
COHEN,
The final washed preparation studies.
RUSKA
AND
RUSKA
was used for the electron
microscope
Electron Microscopy The fluid was removed and the tubes were placed horizontally with the cell sheet on the uppermost side of the tube. Approximately 1 ml of aqueous 2% osmium tetroxide solution was introduced into the tube beneath the cells (Fig. l), which were fixed in the vapor for 10 to 15 minutes. Residual osmium tetroxide solution was washed three times from the glass wall with isotonic buffer. The fixed cells were dehydrated with graduated alcohols in 15 minutes and covered with butyl methacrylate for W hour (three changes). They were then immersed in butyl methacrylate with 2 % Lucid01 for 20 minut,es and embedded in pre-
FIG.
3. Cyto-hemadsorption.
Magnification:
X 16,250.
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
6%
FIG. 4. Cyto-hemadsorption and virus-hemadsorption. Upper red cell is adsorbed by contact of cell membranes. At lower right, red cell is attached to culture cell by interaction of virus filaments in stats nascendi (arrows). Magnification: X 22,500.
polymerized butyl methacrylate. Before final polymerization under nitrogen, the tubes were placed almost horizontally so that the cell sheet was covered with a wedge-shaped layer of embedding medium with a maximum thickness of 5 mm. To remove the plastic from the glass tube, t’he closed t’ubes were placed for a few minutes in a dry-ice-alcohol mixture. This causes t,he plastic to separate from the glass wall and the tube breaks with a loud crack. The area of plastic cont’aining t,he cells
694
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
FIG. 5. Cyto-hemadsorption, virus-hemadsorption, and hemagglutination. Border zones of two tissue culture cells. The upper cell shows cyto- and virus-hemadsorption. Along the lower cell virus-hemadsorption and hemagglutination are mediated by virus filaments. Magnification: X 10,000.
was cut into slices of l-3 mm thickness and the slices were cemented on top of a conventional plastic block to hold the specimen in the microtome. The surface to be cut was reduced to $$ mm >( l-l% mm (Fig. 1). A Porter-Blum microtome with a diamond knife (obtained from the Venezuelan Institute for Scientific Investigation, Caracas) was used for sectioning, and a Siemens Elmiskop I for the electron microscopy. Figures 2-8 were made of a tissue culture inoculated with about lo6
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
695
50 % egg infective doses of the influenza virus and tested for hemadsorption after 24 hours’ incubation. Figure 9 was from a culture incubated for 6 hours after infection. RESULTS
Electron microscopy revealed that red cells were adsorbed to virusinfected monkey kidney cells by t,wo different processes. In one case guinea pig red cells were frequently tightly bound to the surface of
FIG. 6. Virlls-hemadsorption and hemagglutination culture cell. hlaguification: X 10,000.
along
surface
of kidney
696
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
FIG. 7. Virus particles adsorbed on the red cells near surface of kidney culture cell. Red cells are slightly compressed. Note the numerous filamentous, tubular forms of virus and their close proximity along the surface of red cells. Magnification: X 11,250.
infected tissue culture cells in areas where no filamentous processes were seen (Figs. 2 and 3). The red cells were flattened onto the influenza virushost cell and even appeared in some areas to be drawn into the host cell or to have caused a depression or protrusion of the cell surface. Such attachment was direct between the red cell and monkey kidney cell and involved the cell membrane only, wit,h no intermediary virus. In the second type of attachment, red cells were adsorbed to the virus filaments
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZS
VIRUS
697
which protruded from the infected monkey kidney cells. Both types of at’tachment could be seen on some cells (Figs. 4 and 5). In some sections hemagglutination could be seen as red cells with virus bridges between them; here, the red cells were separated by a distance of up to 1 P. A similar dist’ance separated red cells from host cells when t’hey were attached by virus filaments (Figs. 5-7). Control sections of red cells agglutinated by the same strain of influenza virus in allantoic fluid also showed virus bridges between the cells.
FIG. 8. Beginning of virus release and virus-hemadsorption. Note that cytoplasmic and nuclear structures of the host cell are better preserved than in cells which show cyto-hemadsorption. Magnification: X 11,250.
698
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
FIG. 9. Kidney culture cell of almost normal appearance 6 hours after in. fection of the culture. Note the dense mitochondria (m) and the parallel membranes of the endoplasmic reticulum (er). A Golgi zone (G) is visible in the center of the cytoplasm. Magnification: X 11,250.
The two forms of hemadsorption described differ from each other in respect to the presence or absence of an intermediary agent-in this case virus. Both forms differ from hemagglutination in which only one cell type is involved (Fig. 10). We have used the term hemagglutination in the usual sense, restricted to the binding together of red cells by virus. For hemadsorption of the direct type involving red cells attached intimately to the host cell membrane without visible virus we propose the
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
6’39
term “cyto-hemadsorption.” The indirect type, where virus filaments protruding as part of the host cell are attached to a red cell, we have These two forms of attachment can be called “virus-hemadsorption.” distinguished only by electron microscopy. The structure of host cells which showed cyto-hemadsorption was always altered. The cytoplasm contained only a few small mitoc+hondria with a matrix of low density. Vesicles of varying size (50 rnp to several P) were seen and small granules att’ached to the membranes of the vesicles 01 scattered in the rytoplasmic matrix. The endoplasmic reticulum in its normal form with close spaces between parallel membranes had disappeared (compare lcigs. 2-8 with Fig. 9). The nuclear double membrane was preserved but, frequently interrupted, and the inner structure of t,he nuclei was abnormally loose (Figs. 2, 3, and 5). The structure and size of cells several hours after infection indicated considerable swelling (Fig. 9). Culture cells which showed virus-hemadsorption along their surface wit)h additional hemagglutination were in the state of virus release; cytoplasmic and nuclear structures in these had undergone similar but less marked changes than those seen in cells with cyto-hemadsorption (Fig. 8).
HEMAGGLUTINATION
VIRUS-HEMADSORPTION FIG. 10. Diagram
of different
forms
of hemadsorption.
700
HOTCHIN,
COHEN,
RUSKA
AND
RUSKA
The extracellular virus particles appeared either as rings with a diameter of approximately 70 rnF or as tubes of the same diameter and a length up to 2 P. In many instances virus particles were crowded upon the red cell surface so tightly as to indicate limitation only for geometrical reasons (Fig. 7). Thus, it appears that a single red cell could adsorb a very large number of spherical virus particles, possibly in the order of 10,000. DISCUSSION
Hemadsorption reactions with influenza virus in tissue culture have been found to be highly sensitive and specific in accordance with the observations of Shelokov et al. (1958). The electron micrographs suggest that after infection with influenza virus the host cell surface undergoes a change in its immunologically specific structure, rendering the surface capable of forming an attachment to red cell membranes analogous to that shown by mature infectious virus particles. This adsorption occurs only at the surface of virus-infected cells, and is absent in uninoculated controls. The effect is observed where few or no virus filaments of the type described by Morgan et al. (1956) and Bang and Isaacs (1957) are seen at the cell surface. In this respect the host cell behaves as if it were a single large virus particle. Such a change in the structure of the surface of the host cell, in which it becomes antigenically similar to the structure of the surface of the virus, may explain how the virus gains its surface immunological specificity during the last step of its formation as an extrusion of material into a tube of protruding cell membrane. This hypothesis is consistent with reported findings (Knight, 1946; Smith et al., 1955) that influenza virus contains antigens belonging to the host cell in addition to those specifically viral. The virus particle may be in fact enclosed by a membrane, which is essentially the same as that of the altered infected cell. It is possible that after infecting the cell the virus behaves as a cytoplasmic gene, the presence of which is reflected as a phenotypic change in the host which can be detected by the ability of the host cell membrane to behave in a manner similar to that of the virus membrane. The concept that the myxoviruses induce a phenotypic change in the surface of the infected host cell is being studied further. The electron microscopical findings also suggest that all parts of the surface of the red cell are capable of specific attachment to the infected host cell. Furthermore, virus particles have been seen crowded upon the surface of red cells. These observations indicate that the number of
HEMADSORPTION
IN
CULTURES
INFECTED
WITH
INFLUENZA
VIRUS
701
available sites of specific attachment for virus particles may be very great, in sharp contrast to the estimate of about 300 made by Sagik et al. (1954) ; more recently this figure has been considered by Tyrrell and Valentine (1957) to be an underestimation. REFERENCES BANG, F. B., and ISAACS, A. (1957). Morphological
aspects of virus cell relationmumps and Newcastle (Mysouirt~s). In Ciba Foundation Symposium on the Nature of Viruses (G. E. W. Wolstenholme and E. C. P. Millar, eds.), pp. 249-262. Little, Brown and Company, Boston. BODIAN, I). (1956). Simplified method of dispersion of monkey kidney cells with trypsin. Virology 2, 575-576. EAGLE, H. (1955). Utilization of dipeptides by mammalian cells in tissue cult.ure. ships in influenza,
Proc. Sot. Exptl. Biol. Med. 89, 96-99. EAGLE, H., OYAMA, V. I., LEVY, M., and FREEMAN, A. (1956). Myo-inositol
as an essential growth factor for normal and malignant human cells in tissue culture. Science X%3,845-847. KNIGHT, C. A. (1946). Precipitin reactions of highly purified influenza viruses and related materials. J. Exptl. Med. 83, 281-294. MORGAN, C., ROSE, H. M., and MOORE, D. H. (1956). Structure and development of viruses observed in the electron microscope. III. Influenza virus. J. Erptl. ‘2l’ed. 104, 171-182. SAGIK, B.. PUCK, T., and LEVINE, S. (1954). Quantitative aspects of the spontaneous elution of influenza virus from red cells. J. Exptl. Med. 99, 251-260. SHELOKOV, A., VOGEL, J. E., and CHI, L. (1958). Hemadsorption (adsorpt,ionhemagglutination) test for viral agent,s in tissue culture with special reference to influenza. Proc. Sot. Exptl. Biol. Med. 97, 802-809. SMITII, W., BELYAVIN, G., and SHEFFIELD, F. W. (1955). The host-tissue component of influenza viruses. Proc. Roy. Sot. B143, 504-522. TYRRELL, I). A. J., and VALENTINE, R. C. (1957). The assay of influenza virus part,icles by haemagglutination and electron microscopy. J. Gen. Microbial. 16, 668-675. VOGEL, J., and SHELOKOV, A. (1957). Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126, 358-359. YOUNCNER, J. S. (1954). Monolayer tissue cultures. I. Preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells. hoc. Sot. Exptl.
Biol.
Med. 85, 202-205.