- Email: [email protected]
Hemolysis and the release of potassium from cells by Newcastle disease virus (NDV)
VIROLOGY
12, 540-552 (1960)
Hemolysis
National
and the Release of Potassium from by Newcastle Disease Virus (NDV)
Institute
jor
Medical
Research,
London,
N.W.
Cells
Y, England
Accepted August 22, 1960 Hemolysis by NDV was preceded by a rapid release of potassium from the cells and by cell swelling. The potassium release reflected an increased cation permeability of the cell due to the action of the virus. This caused an over-all rise in the internal ionic concentration and osmotic pressure due to the influx of ions from the medium, and led to cell swelling and lysis. Antiserum abolished the reaction of the virus with the cell. Hemolysis was inhibited also by substances that prevented cell swelling but did not affect the initial action of the virus. Cell swelling and hemolysis were reversibly inhibited by albumin, which raised the external osmotic pressure, and by calcium, which probably reduced the cation permeability of the cells. Phloridzin inhibited hemolysis irreversibly by diminishing cell swelling in an unexplained manner. Hemolytic viruses also released potassium and protein from HeLa cells and ascites tumor cells. No detectable release of potassium occurred in the presence of other viruses that are known to infect these cells. The release of large amounts of potassium is therefore due to the hemolytic action of a virus and is not associated with the initiation of infection. INTRODUCTION
The release of intracellular material from bacteria during invasion by bacteriophage (Puck and Lee, 1954) suggests the possibility that a similar loss of material may occur from animal cells at the moment of virus infection. Most animal cells contain potassium in relatively high concentration, and the release of this ion on the addition of virus should be readily detectable. Moreover, the fact that red cell lysis under various conditions is preceded by a loss of potassium from t,he cell (e.g., Ponder, 1948) suggests that a similar potassium loss may be caused by hemolytic viruses. Many hemolytic agents act primarily by increasing the permeability of the red cell membrane to cations (Davson and Ponder, 1940; Wilbrandt, 1941), and the lysis of ascites tumor cells 1 Present address: Pharmacology Medicine, New Haven, Connect,icut.
Department, 540
Yale
University
School
of
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
SDV
541
by antiserum has been attributed to the same primary action (Green et al., 1959). Owing to the high intracellular concentration of macromolecules the cell must maintain the total internal inorganic ion concentrat’ion lower than outside in order not to develop an excessive internal osmotic pressure. This is normally achieved by a relative impermeability of the cell membrane to cations. If free diffusion of inorganic ions can occur across the cell membrane, then the total internal ionic concentration will increase owing to the influx of ions from the medium, although potassium will leak out if its intracellular concentration is higher than its concentration outside. The resulting increase in the osmot,ic pressure of the cytoplasm is responsible for the uptake of water and swelling of the cell which may proceed to the point where high molecular weight material like hemoglobin can escape through the cell membrane. In the present paper it is shown that the ability of a virus to release potassium from both erythrocytes and other cells is associated with hemolytic activity and is therefore not necessarily related to the initiation of infection. Hemolysis can be explained as the consequence of a primary reaction between virus and red cell which is accompanied by potassium release. MATERIALS
AND METHODS
Virus Stocks The following myxoviruses were cultured in the allantoic cavity of eggs: influenza A, strain MEL; a strain of fowl plague virus received from Dr. H. G. Pereira, which was adapted to human lung (Chaproniere and .Pereira, 1955) and subsequently to HeLa cells; the “Herts” (weakly (h emolytic) strains of KDV; and the hemolytic) and “Bonaparte” Sendai strain of parainfluenza 1. These viruses were used either in the form of infected allantoic fluid or after partial purification by adsorption and elution from fowl red blood cells. Yo differences were noted between the actions of these two t,ypes of preparation. Myxovirus hemagglutinin (HA) was titrated by twofold dilutions of 0.25-ml unit volumes and using a final 0.25 % fowl red cell concentration. The HA titer is expressed as HA units per 0.25 ml. Vaccinia virus was purified from infected chorioallantoic membranes by fluorocarbon treatment and centrifugation (Gessler et al., 1956) and had a titer of approximately log HeLa cell infective doses per milliliter. Adenovirus type 5 was partially purified from infected HeLa cells by fluorocarbon treatment and digestion with trypsin (Pereira and Valen-
542
liLEiW’ERER
tine, 1958; Pereira, 1958) and had a titer of approximately lo9 HeLa cell infective doses per milliliter. Encephalomyocarditis virus, kindly supplied by Mr. E. RI. Martin, was a 3000 g supernatant from infected ascitIes c*ell cult,ures (Sanders pt al., 1958) and contained approximately 10y ascites cell infective doses per milliliter. Before use all virus preparat,ions were dialyzed for at least 4 hours at 4” against the medium in which the effect on cells was t,ested. Antiserum Immune serum kindly supplied by virus was studied, units per HA unit
from convalescent’ fowls infected with IXDV was Mr. l?. D. Asplin. When the effect of antiserum on virus was mixed with antiserum (3 HA neutralizing virus) for 10 minutes at’ 37” before use.
Cells Fowl erythrocytes from heparinized blood were washed three times with saline and stored at 4” until required. HeLa cells were cultured as monolayers in S-ounce flat medicine bottles in Gey’s solution containing 5 % rabbit serum and 0.5 % lact’albumin hydrolyzate. The cultures, each cont,aining about 20 million cells, were washed t’wice at 37” before use with the same medium as that in which the effect of virus was tested. Krebs-2 ascites tumor cells maintained by passage in mice were kindly provided by Mr. E. M. Martin. Ascitic fluid was drawn direct into Gey’s solution buffered with 0.01 M 2-amino-2-hydroxymethylpropane1,3-diol (Tris) at pH 7.4. The cells were separated at once by centrifugation for 1 minute at 100 g and washed twice in saline containing 0.01 M Tris pH 7.4 and 0.1% glucose. The cells were then used immediately for experiment’s in which the action of virus was studied. Znteraction of Virus and Cells The action of virus on red blood cells was studied in saline containing 0.01 M sodium phosphate pH 7.4. A 10 % suspension of cells was mixed at 37” with 10 volumes of an appropriate dilution of virus. Samples were taken at intervals, diluted with 2 volumes of saline at 20”, and centrifuged 2 minutes at 600 g. The supernatant was taken for measurement of hemoglobin (optical density at 578 rnp, l-cm light path) and for estimation of potassium by flame photometry. The values are expressed as a percentage of t’he amounts released by a similar dilution of red cells in dist,illed wat,er (“100 % lysis”).
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
543
Inhibitors of hemolysis were dissolved in the test medium and the pH was adjusted to pH 7.4 with the glass electrode. The action of calcium was studied in saline containing 0.01 M Tris pH 7.4, and phosphate and citrate were omitted. In experiments with inhibitors, samples were centrifuged without prior dilution in saline. Cell volumes were determined by transferring the cells from samples centrifuged at 600 g t’o hematocrit tubes with 5 ~1 calibrat,ions and centrifuging to constant volume (20 minutes, 2000 g). The action of virus on HeLa cells was studied either in saline containing 0.01 M phosphate pH 7.4 or in saline containing 0.01 M Tris pH 7.4, and calcium and magnesium ions at t,he same concentration as in Gey’s solution (approximately 1.5 and 1.3 m&J, respectively). Virus in 10 ml medium at 37” was added to the washed monolayers, which were then incubated at 37” on a rocker performing three vertical oscillations per minute about a horizontal axis. Samples of the medium were withdrawn at intervals and centrifuged successively at 200 g and 1500 g to remove detached cells and cell debris, respectively. The supernatant was taken for estimation of pot’assium and of protein (optical density at 280 mp, l-cm light pat,h). The values for potassium are expressed as a percentage of the amount of potassium released in distilled water. Values for protein are corrected for that added initially with the virus inoculum. The action of virus on ascites cells was studied in saline containing 0.01 M Tris pH 7.4 and 0.1% glucose. A 10 % suspension of cells (cont,aining approximately 20 million cells per milliliter) was mixed wit,h 5 volumes of virus suspension at. 37” and samples were withdrawn at intervals. After centrifugation (2 minutes at 200 g) 1 ml supernatant was diluted with 2 ml medium and centrifuged again for 2 minutes at 1500 g to remove cell debris before estimating protein (opt’ical density at 280 rnp) and potassium. Potassium release is expressed as a percentage of t,he amount of potassium released in distilled water. Since large amounts of potassium were released from the ascites cells during preparative manipulations the release of potassium was t#aken as the increase over a “zero time” value measured by spinning down the cells immediat#ely after mixing them with t’he medium at) 37”. RESULTS
Potassium Release from Erythrocytes The hemolytic “Bonaparte” strain of NDV at a titer of 50 HA units caused about 90% hemolysis of fowl erythrocytes in 45 minutes at 37”.
514
KLEMPERER
Figure 1 shows that hemolysis was preceded by a release of potassium from the cells which was completed within 5 minut~es of adding virus, whereas hemoglobin release continued for over 30 minut,es. E’urthermore, potassium release and hemolysis were both diminished at 22” or when lower concentrations of virus were used. The weakly hemolytic “Herts” &rain of NDV also released much less potassium than did the “Bonaparte” strain. In other experiments it’ was found that Sendai virus fell between these two strains in hemolyt’ic activity, a biter of 50 HA units causing about 40% hemolysis in 45 minutes and 70% potassium release. On the other hand fowl plague virus and influenza A strain MEL at, titers up t,o 300 HA units caused neither hemolysis nor potassium release. These data suggest that hemolysis was related to an initial reaction of the virus with the cell which causes a release of potassium. The latter probably reflected an increased permeability of the cell membrane. As outlined in the Introduction an increased permeability to ions is also a primary event in other types of hemolysis and can by itself account for the release of hemoglobin since it permits an increase in the internal ionic concentration with consequent osmotic swelling which leads to lysis.
FIG. 1. Potassium and hemoglobin release from fowl red blood cells. Mixtures of 1O7ored cells with 10 volumes of virus in phosphate-buffered citrate-saline were incubated at 37”, except where otherwise indicated (22”). HA titers refer to the final concentration of “Bonaparte” strain of NIIV. The “Herts” strain was used at a final titer of 50 HA units
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
TABLE
FROM
CELLS
BY
545
NDV
1
CELL SWELLING AND HEMOLYSIS DUE TO NDV”
Condition
of test
Control Virus Virus Virus Virus Virus Virus
+ + + + +
antiserum 3Oa/n albumin 2Ooj, albumin 0.01 M calcium 0.001 M phloridzin
Total cell volume bl)
Ghosts (‘%)
Hemoglobin released ($Zoof total)
30
0
3
65 30 30 45 40 50
70 0 0 0 0 20
80 3 G 8 10 35
a Fowl red cells and NDV (“Bonaparte” strain, titer 50 HA units) were mixed at 37” under conditions as in Fig. 1, except that the effect of calcium was studied in Tris-buffered saline. In the absence of inhibitor the action of virus was the same in both media. After 15 minutes’ incubation 4-ml samples were centrifuged and the cells were then transferred and packed in hematocrit tubes. Total cell volumes (cells plus ghosts) are correct only to the nearest 5 pl. The volume of control cells was unaffected by the presence of inhibitors.
Swelling of the cells as would be expected if virus hemolysis was the consequence of an increased permeability to ions was in fact observed in cells treated with NDV (Table 1). Moreover the swelling preceded lysis. Thus in other experiments the t,otal volume of erythrocytes plus ghosts when sampled between 5 and 45 minutes aft,er adding virus was always about twice that of control cells, while the proportion of ghosts increased from about 30-40 % at 5 minutes to over 80 % at 45 minutes. A study of the effect of hemolysis inhibitors provides evidence that swelling and hemolysis are a consequence of the initial action of the virus which causes potassium release. Inhibition
of Hemolysis
Immune serum, which is known to abolish the hemolytic action of NDV (Burnet and Lind, 1950), also prevented cell swelling (Table 1) and potassium release. Antiserum therefore prevented all reactions of the virus which appear to be connected with lysis. In contrast, other inhibitors of hemolysis shown in Table 1 and Fig. 2 diminished cell swelling without preventing the initial action of the virus that is associated with potassium release. Figure 2 shows that 30% bovine plasma albumin inhibited hemolysis without affecting the potassium release by SDV. In the presence of 30 %
NO INHIBITOR,
loor / I?---- u
-&~O%%N~L~"ii~ 0 NO INHIBITOR
Cd++ a ,_*_-------
FIG. 2. Effect of inhibitors of hemolysis. Hemoglobin (continuous lines) and potassium (interrupted lines) release under the conditions shown in Table 1, except that the final concentration of bovine plasma albumin was 30y0. Hemoglobin and potassium release from control cells was not significantly affected by any inhibitor.
albumin the cells did not swell on the addition of virus (Table 1) but could be shown in other experiments to swell and lyse if subsequent,ly resuspended in a medium without albumin. Albumin would prevent the lysis of cells made freely permeable to inorganic ions by t,he action of virus, if it did not itself penetrate the cell membrane but exerted an external osmotic pressure to balance that due t’o protein inside t’he cell. In the presence of 20% albumin lysis was inhibited but, as expected if 30% albumin prevented swelling by an osmotic action, the cells swelled approximately 1.5 times, which would be sufficient to lower the internal concentration of non-penetrating molecules to two-thirds normal. The slight hemolysis which persisted even in the absence of swelling may have been due t’o an increased suscept,ibility of the cells to mechanical damage. Calcium is known to inhibit hemolysis by h’DV reversibly (Burnet and Lind, 1950). This ion also decreases the permeability of red cells to both potassium and sodium under certain conditions (Kahn, 1958;
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
547
Bolingbroke and Maizels, 1959), and for this reason it prevents the lysis of the erythrocytes of certain species in saline (Maizels, 1956). As shown in Table 1, calcium also inhibited the swelling of red cells in the presence of virus. This would be expected if calcium diminished the cation permeability of the cell and the consequent rise of intracellular osmotic pressure. The same action could account for the inhibition of potassium release by calcium shown in Fig. 2. This stabilizing effect of calcium on cells made potentially permeable by virus action is suggested also by other experiments in which the cells were resuspended in calcium-free medium after virus had acted for 5 minutes or longer in the presence of calcium. Such cells promptly lost their remaining potassium and began to lyse. These experiments do not however exclude the possibility that potassium loss and lysis were completed t,hrough the action of virus which remained adsorbed to the cells. On the other hand, the rapid completion of potassium release in the absence of calcium, even when less than 100% potassium was released (Fig. I) suggests that no further initial action by t,he virus can occur aft’er 5 minutes. Phloridzin, which is known, to inhibit the lysis of red cells by antiserum (Rodriguez and Osler, 1958) also diminished virus hemolysis at concentrations down to 1O-4 M. The release of potassium by virus was unaffect,ed in the presence of phloridzin (Fig. 2) whereas cell swelling was inhibit,ed (Table I), suggesting that phloridzin did not prevent the primary action of t)he virus but stabilized the cells against swelling. That the action of phloridzin is not necessarily directed against any particular type of hemolytic agent is suggested also by other experiments in which the lysis of fowl cells by hypotonic saline was reduced by 1O-3 ,W phloridzin. In contrast to the action of albumin and of calcium, the inhibition of virus hemolysis by phloridzin was irreversible, since hemolysis was not accelerated when t’he cells were resuspended in phloridzin-free medium. This was probably not, due to an irreversible combination of phloridzin with the red cell or the virus since, if either of these was pretreat,ed with phloridzin at 37”, hemolysis in a subsequent test was unaffected if phloridzin was first removed by washing or dialysis. Potassium
Release from
HeLa
Cells
HeLa cell monolayers showed a high rate of spontaneous potassium loss in buffered saline, but this was diminished by the presence of calcium and magnesium at the concentrations used in Gey’s solution. Figure 3A shows that under these conditions the addition of NDV
OY 0
’ 7
I
I
20
45
0.15
FIG. 3. Potassium and protein release from HeLa cells. Potassium (continuous lines) and protein (interrupted lines) released from HeLa cell monolayers at 37’. A. Potassium release in Tris-buffered saline containing calcium and magnesium ions; NDV was used at HA titers of 10 and 100 units, influenza A strain MEL and fowl plague virus (FPV), at HA t i t ers of 300 units. B. NDV was used at a titer of 100 HA units in phosphate-buffered saline. Protein was measured as optical density (OD) a.t 280 mp. 548
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
549
(L‘Bonaparte” strain) at titers from 10 to 100 HA units increased the rate of potassium release. In other experiments this was abolished by immune serum and was approximately 50% inhibited by 0.01 ,!l1 calcium. Figure 3A also shows that fowl plague virus and influenza A strain MEL did not increase the rat,e of potassium release at titers up to 300 HA units, even though the fowl plague virus was a strain adapted to grow in HeLa cells and influenza A virus strains are known to be capable of initiating infection in HeLa cells (Henle et al., 1955). In other experiments potassium release was also not affected by adenovirus type 5 or vaccinia at concentrations of 1O8 infective doses per milliliter. Under these conditions it is likely t,hat virus would be adsorbed to a high proportion of the cells during the first few minutes of incubation, although the init#iation of infect,ion need not necessarily follow adsorption of the virus. HeLa cells showed a spontaneous release of protein as well as of potassium. The tendency to leak intracellular protein, especially in incomplete media, has often been noted in tumor cells (e.g., Macdonald, 1959) and in t’issue culture cells (e.g., Matzelt and Homann, 1958). This spontaneous release was reduced by calcium, which is also known to diminish the loss of protein from tissue slices in artificial media (e.g., Aebi, 1950). In the presence of calcium no effect of virus on protein loss could be demonstrated, but in calcium-free medium NDV increased the rate of spontaneous protein release from the cells as is shown by an increased absorption at 280 rnp in Fig. 3B. Potassium
Release from Ascites Cells
Krebs-2 ascites tumor cells resembled HeLa cells in spontaneously releasing both potassium and protein. In the absence of calcium KDV (“Bonaparte” strain) at a titer of 100 HA units released 100% potassium within 15 minutes and increased the rate of protein release (Fig. 4). Calcium, even at 0.0015 M, considerably inhibited potassium release and prevented an increased release of protein by the virus. The resemblance to hemolysis of this protein release under the influence of virus is further suggested by the finding that after 15 minut)es, when the potassium release was complet,e, the cell volume was about 1.5 times that of t,he controls. Microscopic examinat>ion also showed a higher proportion of larger cells and more cells wit,h faint, or blurred out,lines in the virus-treated sample. In other experiments Sendai virus at, a titer of 300 HA unitIs released only about8 80% potassium in I5 minutes. On t,he other hand,
550
RLICMPERER
influenza A strain MEL at, a t,iter of 300 HA units did not increase the potassium release from the cells although it is likely that ascites cells can be infected by influenza A strains (e.g., Ackermann and Kurtz, 1952). Potassium release was also not affected by encephalomyocarditis virus (2 X lo8 infective doses per milliliter) alt,hough this virus is known t’o grow in t’hese cells and would adsorb to t,hem sufficient)ly rapidly to make initiation of infect,ion possible in pract,irally all of the cells in the first few minutes of incubation (Sanders rt aZ., 1958).
Burnet and Lind (1950) have concluded that hemolysis by NDV is due to an initiating reaction which is completed long before hemolysis reaches its final value. This reaction may be the same as the initial action of the virus now described which caused a breakdown of the relative impermeability of red cells to cations. Thus, as reflected in the rapid release of potassium, t,his breakdown was completed within 5 minutes at 37”, and moreover it could by itself account for lysis of IO0
K+ ND”’
FIG. 4. Potassium and protein release from ascites tumor potassium (continuous lines) and protein (interrupted lines) at cells in Tris-buffered glucose-saline. A 10% suspension of ascites with 5 volumes of NDV at a titer of 100 HA units. Protein was tical density (OD) at 680 m/l.
cells. Release of 37” from ascites cells was mixed measured as op-
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
551
the cells. This is indicated especially by the fact that lysis was reversibly inhibited by albumin and calcium although these substances did not prevent the initial action of the virus. The completion of the initial reaction within 5 minutes at 37” even when less than 100% potassium was released suggests the development of a resistance by the cells to further virus action, such as is found in cells from which the virus receptors have been removed (Burnet and Lind, 1950). This suggests that the durat’ion of the initial react’ion was limited by the rapid receptor-destroying activity of the virus. The subsequent hemolysis was roughly related to the amount of potassium released, since lysis probably depended on the proportion of the cells in which the damage was sufficient to allow swelling to proceed t,o the point where hemoglobin escapes. The release of potassium from HeLa and ascites tumor cells resembled that from red cells in that, it was only caused by hemolytic viruses and was inhibited by antiserum and calcium. Although NDV is probably capable of initiating infect)ion both in HeLa cells (Deinhardt and Henle, 1957) and in ascites tumor cells (Prince and Ginsberg, 1955), this potassium release was probably not related to the entry of virus into the cell since other myxoviruses which also infect these cells caused no potassium release. Similarly the protein release from these cells by NDV, being preceded by potassium release and associated with swelling, was probably due to the same action as that which causes hemolysis. This is in agreement with the conclusion of Henle et al. (1954) that> the rapid cytolyt,ic action of mumps and Newcastle disease viruses on HeLa cells is due to the hemolytic action rather than t’he infect’ive propert’y of these viruses. ACKNOWLEDGMENT I am grateful
to Mr. A. 0. Charles
for technical
assistance.
REFERENCES ACKERMANN, W. W., and KURTZ, H. (1952). A new host-virus system. Proc. Sot. Exptl. Biol. Med. 81, 421-423. AEBI, H. (1950). Kationenmilieu und Gewehsatmung. Helv. Physiol. Acta 8, 525543. BOLINGBROKE, V., and MAIZELS, M. (1959). Calcium ions and the permeability of human erythrocytes. J. Physiol. (London) 149, 563-585. BURNET, F. M., and LIND, P. E. (1950). Haemolysis by Newcastle Disease Virus: IT. General character of haemolytic action. A mtralian J. Exptl. Biol. 28.129-150. CHAPRONIERE, D. M., and PEREIRA, H. G. (1955). Propagation of fowl plague and
552
KLEMPERER
of Newcastle disease virus in cultures of embryonic human lung. H~it. J. Ezptl. Pathol. 36, 607-610. DAVSON, H., and PONDER: E. (1940). Photodynamically induced cation permeability and its relation to haemolysis. J. (‘ellvlar f”omp. Physiol. 16, 67-74. DEINHARDT, F., and HENLE, G. (1957). Studies on the viral spectra of tissue cuture lines of human cells. J. Zrrln~unol. 79, 6@67. GESSLER, A. E., BENDER, C. E., and PARKIXSON, M. C. (1956). A rapid new method for isolating viruses by selective fluorocarbon deproteinization. Trans. X. Y. Acad. Sci. 16, 701-703. GREEN, H., BARROW, P., and GOLDBERG, B. (1959). Effect of antibody and complement on permeability control in ascites tumour cells and erythrocytes. J. Exptl. Med. 110, 699-713. HENLE, G., DEINHARDT, F., and GIRARDI, A. (1954). Cytolytic effects of mumps virus in tissue cult,ures of epithelial cells. Proc. Sot. Exptl. Biol. Med. 87. 386393. HENLE, G., GIRARDI, A., and HENLE, W. (1955). A non-transmissible cytopathic effect of influenza virus in tissue culture accompanied by formation of noninfectious haemagglutinins. J. Exptl. Med. 101, 2541. KAHN, J. B. (1958). Relations between calcium and potassium transfer in human erythrocytes. J. Pharmacol. Exptl. Therap. 123, 263-268. MACDONALD, K. (1959). The release of soluble proteins by ascites tumour cells. Biochim. et Biophys. Acta 36, 543-545. MAIZELS, M. (1956). Sodium transfer in tortoise erythrocytes. J. Physiol. (London) 132, 414-441. MATZELT, D., and HOMANN, J. (1958). Das Verhalten glykolytischer Enzymaktivitiiten in Gewebekulturen vor und nach Beimpfung mit Virus. Biochem. Z. 330, 245-259. PEREIRA, H. G. (1958). A protein factor responsible for the early cytopathic effect of adenoviruses. virology 6, 601-611. PEREIRA, H. G., and VALENTINE, R. C. (1958). Infectivity titrations and particle counts of adenovirus type 5. J. Gen. Microbial. 19, 178-181. PONDER, E. (1948). Haemolysis and Related Phenomena. Churchill, London. PRINCE, A. M., and GINSBERG, H. G. (1955). Immunohistochemical studies on the interaction between Ehrlich ascites tumor cells and Newcastle disease virus. J. Exptl. Med. 106, 177-187. PUCK, T. T., and LEE, H. H. (1954). Mechanism of cell wall penetration by viruses: I. An increase in host cell permeability induced by bacteriophage infection. J. Exptl. Med. 99, 481494. RODRIGUEZ, E., and OSLER, A. G. (1958). Inhibition of immune hemolysis by phlorizin. Federation Proc. 17, 533. SANDERS, F. K., HUPPERT, J., and HUSKING, J. M. (1958). Replication of an animal virus. Symposia Sot. Exptl. Biol. No. 12, 123-137. WILBRANDT, W. (1941). Osmotische Natur sogenannter nicht-osmotischer Haemolysen (kolloidosmotische Haemolyse). Arch. ges. Physiol. Pfltiger’s 246, 22-51.
12, 540-552 (1960)
Hemolysis
National
and the Release of Potassium from by Newcastle Disease Virus (NDV)
Institute
jor
Medical
Research,
London,
N.W.
Cells
Y, England
Accepted August 22, 1960 Hemolysis by NDV was preceded by a rapid release of potassium from the cells and by cell swelling. The potassium release reflected an increased cation permeability of the cell due to the action of the virus. This caused an over-all rise in the internal ionic concentration and osmotic pressure due to the influx of ions from the medium, and led to cell swelling and lysis. Antiserum abolished the reaction of the virus with the cell. Hemolysis was inhibited also by substances that prevented cell swelling but did not affect the initial action of the virus. Cell swelling and hemolysis were reversibly inhibited by albumin, which raised the external osmotic pressure, and by calcium, which probably reduced the cation permeability of the cells. Phloridzin inhibited hemolysis irreversibly by diminishing cell swelling in an unexplained manner. Hemolytic viruses also released potassium and protein from HeLa cells and ascites tumor cells. No detectable release of potassium occurred in the presence of other viruses that are known to infect these cells. The release of large amounts of potassium is therefore due to the hemolytic action of a virus and is not associated with the initiation of infection. INTRODUCTION
The release of intracellular material from bacteria during invasion by bacteriophage (Puck and Lee, 1954) suggests the possibility that a similar loss of material may occur from animal cells at the moment of virus infection. Most animal cells contain potassium in relatively high concentration, and the release of this ion on the addition of virus should be readily detectable. Moreover, the fact that red cell lysis under various conditions is preceded by a loss of potassium from t,he cell (e.g., Ponder, 1948) suggests that a similar potassium loss may be caused by hemolytic viruses. Many hemolytic agents act primarily by increasing the permeability of the red cell membrane to cations (Davson and Ponder, 1940; Wilbrandt, 1941), and the lysis of ascites tumor cells 1 Present address: Pharmacology Medicine, New Haven, Connect,icut.
Department, 540
Yale
University
School
of
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
SDV
541
by antiserum has been attributed to the same primary action (Green et al., 1959). Owing to the high intracellular concentration of macromolecules the cell must maintain the total internal inorganic ion concentrat’ion lower than outside in order not to develop an excessive internal osmotic pressure. This is normally achieved by a relative impermeability of the cell membrane to cations. If free diffusion of inorganic ions can occur across the cell membrane, then the total internal ionic concentration will increase owing to the influx of ions from the medium, although potassium will leak out if its intracellular concentration is higher than its concentration outside. The resulting increase in the osmot,ic pressure of the cytoplasm is responsible for the uptake of water and swelling of the cell which may proceed to the point where high molecular weight material like hemoglobin can escape through the cell membrane. In the present paper it is shown that the ability of a virus to release potassium from both erythrocytes and other cells is associated with hemolytic activity and is therefore not necessarily related to the initiation of infection. Hemolysis can be explained as the consequence of a primary reaction between virus and red cell which is accompanied by potassium release. MATERIALS
AND METHODS
Virus Stocks The following myxoviruses were cultured in the allantoic cavity of eggs: influenza A, strain MEL; a strain of fowl plague virus received from Dr. H. G. Pereira, which was adapted to human lung (Chaproniere and .Pereira, 1955) and subsequently to HeLa cells; the “Herts” (weakly (h emolytic) strains of KDV; and the hemolytic) and “Bonaparte” Sendai strain of parainfluenza 1. These viruses were used either in the form of infected allantoic fluid or after partial purification by adsorption and elution from fowl red blood cells. Yo differences were noted between the actions of these two t,ypes of preparation. Myxovirus hemagglutinin (HA) was titrated by twofold dilutions of 0.25-ml unit volumes and using a final 0.25 % fowl red cell concentration. The HA titer is expressed as HA units per 0.25 ml. Vaccinia virus was purified from infected chorioallantoic membranes by fluorocarbon treatment and centrifugation (Gessler et al., 1956) and had a titer of approximately log HeLa cell infective doses per milliliter. Adenovirus type 5 was partially purified from infected HeLa cells by fluorocarbon treatment and digestion with trypsin (Pereira and Valen-
542
liLEiW’ERER
tine, 1958; Pereira, 1958) and had a titer of approximately lo9 HeLa cell infective doses per milliliter. Encephalomyocarditis virus, kindly supplied by Mr. E. RI. Martin, was a 3000 g supernatant from infected ascitIes c*ell cult,ures (Sanders pt al., 1958) and contained approximately 10y ascites cell infective doses per milliliter. Before use all virus preparat,ions were dialyzed for at least 4 hours at 4” against the medium in which the effect on cells was t,ested. Antiserum Immune serum kindly supplied by virus was studied, units per HA unit
from convalescent’ fowls infected with IXDV was Mr. l?. D. Asplin. When the effect of antiserum on virus was mixed with antiserum (3 HA neutralizing virus) for 10 minutes at’ 37” before use.
Cells Fowl erythrocytes from heparinized blood were washed three times with saline and stored at 4” until required. HeLa cells were cultured as monolayers in S-ounce flat medicine bottles in Gey’s solution containing 5 % rabbit serum and 0.5 % lact’albumin hydrolyzate. The cultures, each cont,aining about 20 million cells, were washed t’wice at 37” before use with the same medium as that in which the effect of virus was tested. Krebs-2 ascites tumor cells maintained by passage in mice were kindly provided by Mr. E. M. Martin. Ascitic fluid was drawn direct into Gey’s solution buffered with 0.01 M 2-amino-2-hydroxymethylpropane1,3-diol (Tris) at pH 7.4. The cells were separated at once by centrifugation for 1 minute at 100 g and washed twice in saline containing 0.01 M Tris pH 7.4 and 0.1% glucose. The cells were then used immediately for experiment’s in which the action of virus was studied. Znteraction of Virus and Cells The action of virus on red blood cells was studied in saline containing 0.01 M sodium phosphate pH 7.4. A 10 % suspension of cells was mixed at 37” with 10 volumes of an appropriate dilution of virus. Samples were taken at intervals, diluted with 2 volumes of saline at 20”, and centrifuged 2 minutes at 600 g. The supernatant was taken for measurement of hemoglobin (optical density at 578 rnp, l-cm light path) and for estimation of potassium by flame photometry. The values are expressed as a percentage of t’he amounts released by a similar dilution of red cells in dist,illed wat,er (“100 % lysis”).
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
543
Inhibitors of hemolysis were dissolved in the test medium and the pH was adjusted to pH 7.4 with the glass electrode. The action of calcium was studied in saline containing 0.01 M Tris pH 7.4, and phosphate and citrate were omitted. In experiments with inhibitors, samples were centrifuged without prior dilution in saline. Cell volumes were determined by transferring the cells from samples centrifuged at 600 g t’o hematocrit tubes with 5 ~1 calibrat,ions and centrifuging to constant volume (20 minutes, 2000 g). The action of virus on HeLa cells was studied either in saline containing 0.01 M phosphate pH 7.4 or in saline containing 0.01 M Tris pH 7.4, and calcium and magnesium ions at t,he same concentration as in Gey’s solution (approximately 1.5 and 1.3 m&J, respectively). Virus in 10 ml medium at 37” was added to the washed monolayers, which were then incubated at 37” on a rocker performing three vertical oscillations per minute about a horizontal axis. Samples of the medium were withdrawn at intervals and centrifuged successively at 200 g and 1500 g to remove detached cells and cell debris, respectively. The supernatant was taken for estimation of pot’assium and of protein (optical density at 280 mp, l-cm light pat,h). The values for potassium are expressed as a percentage of the amount of potassium released in distilled water. Values for protein are corrected for that added initially with the virus inoculum. The action of virus on ascites cells was studied in saline containing 0.01 M Tris pH 7.4 and 0.1% glucose. A 10 % suspension of cells (cont,aining approximately 20 million cells per milliliter) was mixed wit,h 5 volumes of virus suspension at. 37” and samples were withdrawn at intervals. After centrifugation (2 minutes at 200 g) 1 ml supernatant was diluted with 2 ml medium and centrifuged again for 2 minutes at 1500 g to remove cell debris before estimating protein (opt’ical density at 280 rnp) and potassium. Potassium release is expressed as a percentage of t,he amount of potassium released in distilled water. Since large amounts of potassium were released from the ascites cells during preparative manipulations the release of potassium was t#aken as the increase over a “zero time” value measured by spinning down the cells immediat#ely after mixing them with t’he medium at) 37”. RESULTS
Potassium Release from Erythrocytes The hemolytic “Bonaparte” strain of NDV at a titer of 50 HA units caused about 90% hemolysis of fowl erythrocytes in 45 minutes at 37”.
514
KLEMPERER
Figure 1 shows that hemolysis was preceded by a release of potassium from the cells which was completed within 5 minut~es of adding virus, whereas hemoglobin release continued for over 30 minut,es. E’urthermore, potassium release and hemolysis were both diminished at 22” or when lower concentrations of virus were used. The weakly hemolytic “Herts” &rain of NDV also released much less potassium than did the “Bonaparte” strain. In other experiments it’ was found that Sendai virus fell between these two strains in hemolyt’ic activity, a biter of 50 HA units causing about 40% hemolysis in 45 minutes and 70% potassium release. On the other hand fowl plague virus and influenza A strain MEL at, titers up t,o 300 HA units caused neither hemolysis nor potassium release. These data suggest that hemolysis was related to an initial reaction of the virus with the cell which causes a release of potassium. The latter probably reflected an increased permeability of the cell membrane. As outlined in the Introduction an increased permeability to ions is also a primary event in other types of hemolysis and can by itself account for the release of hemoglobin since it permits an increase in the internal ionic concentration with consequent osmotic swelling which leads to lysis.
FIG. 1. Potassium and hemoglobin release from fowl red blood cells. Mixtures of 1O7ored cells with 10 volumes of virus in phosphate-buffered citrate-saline were incubated at 37”, except where otherwise indicated (22”). HA titers refer to the final concentration of “Bonaparte” strain of NIIV. The “Herts” strain was used at a final titer of 50 HA units
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
TABLE
FROM
CELLS
BY
545
NDV
1
CELL SWELLING AND HEMOLYSIS DUE TO NDV”
Condition
of test
Control Virus Virus Virus Virus Virus Virus
+ + + + +
antiserum 3Oa/n albumin 2Ooj, albumin 0.01 M calcium 0.001 M phloridzin
Total cell volume bl)
Ghosts (‘%)
Hemoglobin released ($Zoof total)
30
0
3
65 30 30 45 40 50
70 0 0 0 0 20
80 3 G 8 10 35
a Fowl red cells and NDV (“Bonaparte” strain, titer 50 HA units) were mixed at 37” under conditions as in Fig. 1, except that the effect of calcium was studied in Tris-buffered saline. In the absence of inhibitor the action of virus was the same in both media. After 15 minutes’ incubation 4-ml samples were centrifuged and the cells were then transferred and packed in hematocrit tubes. Total cell volumes (cells plus ghosts) are correct only to the nearest 5 pl. The volume of control cells was unaffected by the presence of inhibitors.
Swelling of the cells as would be expected if virus hemolysis was the consequence of an increased permeability to ions was in fact observed in cells treated with NDV (Table 1). Moreover the swelling preceded lysis. Thus in other experiments the t,otal volume of erythrocytes plus ghosts when sampled between 5 and 45 minutes aft,er adding virus was always about twice that of control cells, while the proportion of ghosts increased from about 30-40 % at 5 minutes to over 80 % at 45 minutes. A study of the effect of hemolysis inhibitors provides evidence that swelling and hemolysis are a consequence of the initial action of the virus which causes potassium release. Inhibition
of Hemolysis
Immune serum, which is known to abolish the hemolytic action of NDV (Burnet and Lind, 1950), also prevented cell swelling (Table 1) and potassium release. Antiserum therefore prevented all reactions of the virus which appear to be connected with lysis. In contrast, other inhibitors of hemolysis shown in Table 1 and Fig. 2 diminished cell swelling without preventing the initial action of the virus that is associated with potassium release. Figure 2 shows that 30% bovine plasma albumin inhibited hemolysis without affecting the potassium release by SDV. In the presence of 30 %
NO INHIBITOR,
loor / I?---- u
-&~O%%N~L~"ii~ 0 NO INHIBITOR
Cd++ a ,_*_-------
FIG. 2. Effect of inhibitors of hemolysis. Hemoglobin (continuous lines) and potassium (interrupted lines) release under the conditions shown in Table 1, except that the final concentration of bovine plasma albumin was 30y0. Hemoglobin and potassium release from control cells was not significantly affected by any inhibitor.
albumin the cells did not swell on the addition of virus (Table 1) but could be shown in other experiments to swell and lyse if subsequent,ly resuspended in a medium without albumin. Albumin would prevent the lysis of cells made freely permeable to inorganic ions by t,he action of virus, if it did not itself penetrate the cell membrane but exerted an external osmotic pressure to balance that due t’o protein inside t’he cell. In the presence of 20% albumin lysis was inhibited but, as expected if 30% albumin prevented swelling by an osmotic action, the cells swelled approximately 1.5 times, which would be sufficient to lower the internal concentration of non-penetrating molecules to two-thirds normal. The slight hemolysis which persisted even in the absence of swelling may have been due t’o an increased suscept,ibility of the cells to mechanical damage. Calcium is known to inhibit hemolysis by h’DV reversibly (Burnet and Lind, 1950). This ion also decreases the permeability of red cells to both potassium and sodium under certain conditions (Kahn, 1958;
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
547
Bolingbroke and Maizels, 1959), and for this reason it prevents the lysis of the erythrocytes of certain species in saline (Maizels, 1956). As shown in Table 1, calcium also inhibited the swelling of red cells in the presence of virus. This would be expected if calcium diminished the cation permeability of the cell and the consequent rise of intracellular osmotic pressure. The same action could account for the inhibition of potassium release by calcium shown in Fig. 2. This stabilizing effect of calcium on cells made potentially permeable by virus action is suggested also by other experiments in which the cells were resuspended in calcium-free medium after virus had acted for 5 minutes or longer in the presence of calcium. Such cells promptly lost their remaining potassium and began to lyse. These experiments do not however exclude the possibility that potassium loss and lysis were completed t,hrough the action of virus which remained adsorbed to the cells. On the other hand, the rapid completion of potassium release in the absence of calcium, even when less than 100% potassium was released (Fig. I) suggests that no further initial action by t,he virus can occur aft’er 5 minutes. Phloridzin, which is known, to inhibit the lysis of red cells by antiserum (Rodriguez and Osler, 1958) also diminished virus hemolysis at concentrations down to 1O-4 M. The release of potassium by virus was unaffect,ed in the presence of phloridzin (Fig. 2) whereas cell swelling was inhibit,ed (Table I), suggesting that phloridzin did not prevent the primary action of t)he virus but stabilized the cells against swelling. That the action of phloridzin is not necessarily directed against any particular type of hemolytic agent is suggested also by other experiments in which the lysis of fowl cells by hypotonic saline was reduced by 1O-3 ,W phloridzin. In contrast to the action of albumin and of calcium, the inhibition of virus hemolysis by phloridzin was irreversible, since hemolysis was not accelerated when t’he cells were resuspended in phloridzin-free medium. This was probably not, due to an irreversible combination of phloridzin with the red cell or the virus since, if either of these was pretreat,ed with phloridzin at 37”, hemolysis in a subsequent test was unaffected if phloridzin was first removed by washing or dialysis. Potassium
Release from
HeLa
Cells
HeLa cell monolayers showed a high rate of spontaneous potassium loss in buffered saline, but this was diminished by the presence of calcium and magnesium at the concentrations used in Gey’s solution. Figure 3A shows that under these conditions the addition of NDV
OY 0
’ 7
I
I
20
45
0.15
FIG. 3. Potassium and protein release from HeLa cells. Potassium (continuous lines) and protein (interrupted lines) released from HeLa cell monolayers at 37’. A. Potassium release in Tris-buffered saline containing calcium and magnesium ions; NDV was used at HA titers of 10 and 100 units, influenza A strain MEL and fowl plague virus (FPV), at HA t i t ers of 300 units. B. NDV was used at a titer of 100 HA units in phosphate-buffered saline. Protein was measured as optical density (OD) a.t 280 mp. 548
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
549
(L‘Bonaparte” strain) at titers from 10 to 100 HA units increased the rate of potassium release. In other experiments this was abolished by immune serum and was approximately 50% inhibited by 0.01 ,!l1 calcium. Figure 3A also shows that fowl plague virus and influenza A strain MEL did not increase the rat,e of potassium release at titers up to 300 HA units, even though the fowl plague virus was a strain adapted to grow in HeLa cells and influenza A virus strains are known to be capable of initiating infection in HeLa cells (Henle et al., 1955). In other experiments potassium release was also not affected by adenovirus type 5 or vaccinia at concentrations of 1O8 infective doses per milliliter. Under these conditions it is likely t,hat virus would be adsorbed to a high proportion of the cells during the first few minutes of incubation, although the init#iation of infect,ion need not necessarily follow adsorption of the virus. HeLa cells showed a spontaneous release of protein as well as of potassium. The tendency to leak intracellular protein, especially in incomplete media, has often been noted in tumor cells (e.g., Macdonald, 1959) and in t’issue culture cells (e.g., Matzelt and Homann, 1958). This spontaneous release was reduced by calcium, which is also known to diminish the loss of protein from tissue slices in artificial media (e.g., Aebi, 1950). In the presence of calcium no effect of virus on protein loss could be demonstrated, but in calcium-free medium NDV increased the rate of spontaneous protein release from the cells as is shown by an increased absorption at 280 rnp in Fig. 3B. Potassium
Release from Ascites Cells
Krebs-2 ascites tumor cells resembled HeLa cells in spontaneously releasing both potassium and protein. In the absence of calcium KDV (“Bonaparte” strain) at a titer of 100 HA units released 100% potassium within 15 minutes and increased the rate of protein release (Fig. 4). Calcium, even at 0.0015 M, considerably inhibited potassium release and prevented an increased release of protein by the virus. The resemblance to hemolysis of this protein release under the influence of virus is further suggested by the finding that after 15 minut)es, when the potassium release was complet,e, the cell volume was about 1.5 times that of t,he controls. Microscopic examinat>ion also showed a higher proportion of larger cells and more cells wit,h faint, or blurred out,lines in the virus-treated sample. In other experiments Sendai virus at, a titer of 300 HA unitIs released only about8 80% potassium in I5 minutes. On t,he other hand,
550
RLICMPERER
influenza A strain MEL at, a t,iter of 300 HA units did not increase the potassium release from the cells although it is likely that ascites cells can be infected by influenza A strains (e.g., Ackermann and Kurtz, 1952). Potassium release was also not affected by encephalomyocarditis virus (2 X lo8 infective doses per milliliter) alt,hough this virus is known t’o grow in t’hese cells and would adsorb to t,hem sufficient)ly rapidly to make initiation of infect,ion possible in pract,irally all of the cells in the first few minutes of incubation (Sanders rt aZ., 1958).
Burnet and Lind (1950) have concluded that hemolysis by NDV is due to an initiating reaction which is completed long before hemolysis reaches its final value. This reaction may be the same as the initial action of the virus now described which caused a breakdown of the relative impermeability of red cells to cations. Thus, as reflected in the rapid release of potassium, t,his breakdown was completed within 5 minutes at 37”, and moreover it could by itself account for lysis of IO0
K+ ND”’
FIG. 4. Potassium and protein release from ascites tumor potassium (continuous lines) and protein (interrupted lines) at cells in Tris-buffered glucose-saline. A 10% suspension of ascites with 5 volumes of NDV at a titer of 100 HA units. Protein was tical density (OD) at 680 m/l.
cells. Release of 37” from ascites cells was mixed measured as op-
HEMOLYSIS
AND
RELEASE
OF POTASSIUM
FROM
CELLS
BY
NDV
551
the cells. This is indicated especially by the fact that lysis was reversibly inhibited by albumin and calcium although these substances did not prevent the initial action of the virus. The completion of the initial reaction within 5 minutes at 37” even when less than 100% potassium was released suggests the development of a resistance by the cells to further virus action, such as is found in cells from which the virus receptors have been removed (Burnet and Lind, 1950). This suggests that the durat’ion of the initial react’ion was limited by the rapid receptor-destroying activity of the virus. The subsequent hemolysis was roughly related to the amount of potassium released, since lysis probably depended on the proportion of the cells in which the damage was sufficient to allow swelling to proceed t,o the point where hemoglobin escapes. The release of potassium from HeLa and ascites tumor cells resembled that from red cells in that, it was only caused by hemolytic viruses and was inhibited by antiserum and calcium. Although NDV is probably capable of initiating infect)ion both in HeLa cells (Deinhardt and Henle, 1957) and in ascites tumor cells (Prince and Ginsberg, 1955), this potassium release was probably not related to the entry of virus into the cell since other myxoviruses which also infect these cells caused no potassium release. Similarly the protein release from these cells by NDV, being preceded by potassium release and associated with swelling, was probably due to the same action as that which causes hemolysis. This is in agreement with the conclusion of Henle et al. (1954) that> the rapid cytolyt,ic action of mumps and Newcastle disease viruses on HeLa cells is due to the hemolytic action rather than t’he infect’ive propert’y of these viruses. ACKNOWLEDGMENT I am grateful
to Mr. A. 0. Charles
for technical
assistance.
REFERENCES ACKERMANN, W. W., and KURTZ, H. (1952). A new host-virus system. Proc. Sot. Exptl. Biol. Med. 81, 421-423. AEBI, H. (1950). Kationenmilieu und Gewehsatmung. Helv. Physiol. Acta 8, 525543. BOLINGBROKE, V., and MAIZELS, M. (1959). Calcium ions and the permeability of human erythrocytes. J. Physiol. (London) 149, 563-585. BURNET, F. M., and LIND, P. E. (1950). Haemolysis by Newcastle Disease Virus: IT. General character of haemolytic action. A mtralian J. Exptl. Biol. 28.129-150. CHAPRONIERE, D. M., and PEREIRA, H. G. (1955). Propagation of fowl plague and
552
KLEMPERER
of Newcastle disease virus in cultures of embryonic human lung. H~it. J. Ezptl. Pathol. 36, 607-610. DAVSON, H., and PONDER: E. (1940). Photodynamically induced cation permeability and its relation to haemolysis. J. (‘ellvlar f”omp. Physiol. 16, 67-74. DEINHARDT, F., and HENLE, G. (1957). Studies on the viral spectra of tissue cuture lines of human cells. J. Zrrln~unol. 79, 6@67. GESSLER, A. E., BENDER, C. E., and PARKIXSON, M. C. (1956). A rapid new method for isolating viruses by selective fluorocarbon deproteinization. Trans. X. Y. Acad. Sci. 16, 701-703. GREEN, H., BARROW, P., and GOLDBERG, B. (1959). Effect of antibody and complement on permeability control in ascites tumour cells and erythrocytes. J. Exptl. Med. 110, 699-713. HENLE, G., DEINHARDT, F., and GIRARDI, A. (1954). Cytolytic effects of mumps virus in tissue cult,ures of epithelial cells. Proc. Sot. Exptl. Biol. Med. 87. 386393. HENLE, G., GIRARDI, A., and HENLE, W. (1955). A non-transmissible cytopathic effect of influenza virus in tissue culture accompanied by formation of noninfectious haemagglutinins. J. Exptl. Med. 101, 2541. KAHN, J. B. (1958). Relations between calcium and potassium transfer in human erythrocytes. J. Pharmacol. Exptl. Therap. 123, 263-268. MACDONALD, K. (1959). The release of soluble proteins by ascites tumour cells. Biochim. et Biophys. Acta 36, 543-545. MAIZELS, M. (1956). Sodium transfer in tortoise erythrocytes. J. Physiol. (London) 132, 414-441. MATZELT, D., and HOMANN, J. (1958). Das Verhalten glykolytischer Enzymaktivitiiten in Gewebekulturen vor und nach Beimpfung mit Virus. Biochem. Z. 330, 245-259. PEREIRA, H. G. (1958). A protein factor responsible for the early cytopathic effect of adenoviruses. virology 6, 601-611. PEREIRA, H. G., and VALENTINE, R. C. (1958). Infectivity titrations and particle counts of adenovirus type 5. J. Gen. Microbial. 19, 178-181. PONDER, E. (1948). Haemolysis and Related Phenomena. Churchill, London. PRINCE, A. M., and GINSBERG, H. G. (1955). Immunohistochemical studies on the interaction between Ehrlich ascites tumor cells and Newcastle disease virus. J. Exptl. Med. 106, 177-187. PUCK, T. T., and LEE, H. H. (1954). Mechanism of cell wall penetration by viruses: I. An increase in host cell permeability induced by bacteriophage infection. J. Exptl. Med. 99, 481494. RODRIGUEZ, E., and OSLER, A. G. (1958). Inhibition of immune hemolysis by phlorizin. Federation Proc. 17, 533. SANDERS, F. K., HUPPERT, J., and HUSKING, J. M. (1958). Replication of an animal virus. Symposia Sot. Exptl. Biol. No. 12, 123-137. WILBRANDT, W. (1941). Osmotische Natur sogenannter nicht-osmotischer Haemolysen (kolloidosmotische Haemolyse). Arch. ges. Physiol. Pfltiger’s 246, 22-51.