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Effect of antiserum on the cell fusion reaction caused by HVJ
22, 39’i‘iog
VIROLOGY
Effect
(1964)
of Antiserum YOSHIO
Department
OKADA, of Preventive
on the Cell KAZUKO
Fusion
YAMADA,
Reaction AND
October
by HVJ’
JUN T,4DOKOR02
Medicine, The Research Institute Osaka University, Osaka, Japan Accepted
Caused
for Microbial
Diseases,
15, 1963
The cell fusion reaction of Ehrlich ascites tumor cells caused by HVJ was completely inhibited by using a large amount. of anti-HVJ serum. However, when diluted antiserum was added to the HVJ sample, the cell fusion reaction was enhanced. In the sample, showing maximal enhancement of the reaction, almost all the cell fusion activity was found in virus flocculates which were sedimentable by low speed centrifugation. When antiserum against other myxoviruses, e.g., NDV or PRS, was mixed with the HVJ sample, virus flocculation also occurred together with enhancement of the cell fusion reaction. Both activities, the HVJ flocculating and the cell fusion-enhancing activities, were lost after absorption with sedimentable components in normal chorioallantoic fluid. The enhancement of the cell fusion reaction with anti-HVJ or antisera against other myxoviruses may be correlated with the formation of virus clumps, in which the cell fusion activity of the participant virus is kept as high as that of the free virus. INTRODUCTION
In 1957, Okada et al. reported the appearance of many giant polynuclear tumor cells within an hour after a large dose of HVJ had been inoculated into the abdominal cavity of a mouse implanted with Ehrlich ascites tumor cells; they showed that the cells agglutinated as a result of virus adsorption and fused together, and that multinucleated giant cells appeared. Subsequently this phenomenon was analyzed in vitro (Okada, 1958, 1962a,b; Okada and Tadokoro, 1962). As a continuation of this work, the effect of anti-HVJ serum on the cell fusion reaction was studied; the results are described in this report. The reaction was completely inhibited by the addition of a sufficiently large titer of the antiserum. But surprisingly, when a small dose of antiserum was added to the HVJ sample, the cell fusion 1 Hemagglutinating virus of Japan, Sendai virus. * Present address : Department of Osaka National Hospital.
synonym
:
Pediatrics, 397
reaction was strikingly enhanced compared with the effect of the virus only. Cowie et al. (1961) reported that exposure of ribosome-bound p-galactosidase to antiserum against P-galactosidase resulted in a three- to sixfold increase in activity. In this report, the enhancement of the cell fusion reaction by virus that has combined with a small amount of antibody is described and discussed. The inhibition caused by a large dose of the antiserum is also reported. MATERIALS
AND
METHODS
I’irus. The egg-adapted Z strain of HVJ was used. The virus was propagated in the allantoic sacs of lo-day-old chick embryos, using a diluted inoculum. The allantoic fluid was centrifuged at 1400 g for 20 minutes. Then the supernatant was centrifuged at 34,850 g for 30 minutes. The sediment was suspended in modified Hanks’ solution and was recent,rifuged at 1400 g for 20 minutes to eliminate aggregates. Cells. Ehrlich hypertetraploid ascites
398
OKADA,
YAMADA,
tumor cells (ETC) were withdrawn from the peritoneal cavity of adult Swiss ddO mice inoculated 7 days previously with 0.1 ml of tenfold diluted tumor-bearing ascites fluid. The cells were washed three or four times with modified Hanks’ solution to remove contaminating red cells and leucocytes and were suspended in the same solution at a concentration of 1.2 X IO7 cells/ml. Anti-HVJ, NDV, and PR8 sera. Aliquots of 1 ml of purified HVJ, Newcastle disease virus (NDV) or PR8 strain of influenza virus (10,000 hemagglutinating units per milliliter), propagated in embryonated eggs, were injected into a rabbit, alternately intravenously and intramuscularly once a week. One week after the third injection, the blood was withdrawn by cardiac puncture and the antisera were incubated at 56°C for 60 minutes. These antisera were supplied by Dr. Y. Hosaka of our laboratory. Also antisera obtained by immunization with mixed samples of viruses and incomplete adjuvant (composed of Arlacel and Drackeol No. 6) were used. Modified Hanks’ solution. Hanks’ balanced salt, solution, without glucose or bicarbonate, was used throughout the work. The pH was adjusted to 7.6 with tris(hybuffer droxymethyl) aminomethane-HCl (0.01 M). Hemagglutination (HA) titration. Salk’s pattern method employing 0.5 ml of 0.5% fowl red blood cells was used with phosphate-buffered saline as the diluent. Under these conditions, one HA unit corresponds to about 2.4 x lo7 virus particles (Okada et al., 1961). Determination
of hemolytic
(HL)
actiu-
One milliliter of the virus sample was mixed with 2 ml of 2% fowl red blood cell suspension. The mixture was kept in the cold for 15 minutes and then washed once with cold phosphate-buffered saline (pH 7.2)) and PBS was added to the original volume. The sample was put in a water bath at 37” for 60 minutes and agitated every 15 minutes. After incubation, the tube was centrifuged at 350 g for 5 minutes. The supernatant fluid was transferred to a calorimeter cuvette and the amount of hemoglobin liberated was determined specity.
AND
TADOKORO
trophotometrically at 540 mp. As a reference PBS was used in place of the virus. When a high concentration of HVJ was used, hemolysis was inhibited as reported by Granoff and Henle (1954) in a NDV-fowl red blood cell system. But if freeze-thawed virus was used, there was no inhibition and the degree of hemolysis increased with the amount of virus added. Moreover, the freeze-thawed virus behaved the same way with anti-HVJ serum as the native virus. For these reasons, only freeze-thawed (2 cycles) virus samples were used when HL activity was to be measured in the experiment. Cell fusion reaction. A 0.5-ml aliquot of the ETC sample containing 1.2 x IO7 cells/ml and an equal volume of virus sample were mixed in a test, tube. The tube was put in an ice bath for 15 minutes to allow cell agglutination. Then the tube was incubated in a water bath at 37” with shaking for 60 minutes. During the incubation the cells in the clumps fused together or became dispersed in the medium ; no cell clumps remained at the end of the incubation. In other words, the decrease in the cell number observed at the end of the reaction corresponded to the degree of cell fusion. After incubation the sample was cooled and mixed by pipette; an aliquot was introduced into a Fuchs-Rosenthal cytometer and photographed with a phase contrast, microscope at low magnification. The cell number of the sample was counted from ten negative plates (under these conditions, about 200 cells were photographed per plate of nonvirus control sample) and the “fusion index” was calculated according to the following formula (Okada and Tadokoro, 1962) : fusion index (F.1.) =
cell number
in nonvirus
cell number
control
in test tube
tube
-
1.0
Determination of the degree of flocculation of the virus with antiserum. A mixture
of virus suspension and trifuged at 1000 g for keeping the sample for and overnight at 4°C.
antiserum was cen20 minutes after 60 minutes at 37” The sediment was
ANTISERUM
EFFECT
389
ON CELL FUSION REACTION
washed with saline, and the protein content was estimated by the method of Lowry et nl. (1951). Agar gel difusion. The double diffusion technique of Ouchterlony was employed. RESULTS
Inhibition of the Cell Fusion Reaction with a Sufficient Titer of Anti-HVJ Serum Aliquots of 0.5 ml of a solution containing 8000 HAU of HVJ per milliliter were mixed in test tubes with an equal volume of a solution containing 1.2 x lo7 cells of ETC per milliliter and kept for 15 minutes at 4”. One milliliter of anti-HVJ serum (with a hemagglutination inhibition titer of 1024 units/ml against 4 HAU of HVJ) was introduced into one of the test tubes during this incubation at 4”, and the tube was incubated at 37” for 60 minutes with shaking. The other test tubes, after being kept at 4” for 15 minutes, were incubated at 37” with shaking, and the same amount of the antiserum was added during the course of the cell fusion reaction at 37”. After completion of the cell fusion reaction (for a total of 60 minutes at 37”), the cell fusion index (F.I.) of these samples was estimated. As indicated in Fig. 1, the cell fusion reaction was completely inhibited only when antiserum was added before incubation at 37” and the antiserum did not affect the reaction in the sample after incubation for 5 minutes or longer at 37”. This experiment showed that the initiation reaction of the cell fusion process with the virus was completed within 5 minutes at 37”. This is compatible with the results of previous studies on the mechanism of cell fusion obtained by electron microscopy, in which it was shown that the virus particles disappeared from the cell surface within 5 minutes on incubation at 37”, and at that time the cell membranes of neighboring cells had already fused together (Okada, 1962a). Enhancement of the Cell Fusion Reaction by a Small Amount of Anti-HVJ Serum One milliliter of anti-HVJ serum, with a titer of 1024 HIU (hemagglutination inhibition units) against 4 HAU of HVJ, was
A-!
0 5 IO
60 min
30
Time of antiserum
addition
FIG. 1. Inhibition of the cell fusion reaction with anti-HVJ serum. Aliquots of 0.5 ml of 8000 HAU of HVJ per milliliter were mixed with an equal volume of 1.2 X 10’ cells of ETC per milliliter in test tubes. One milliliter of undiluted anti-HVJ serum was introduced into the tubes during the cell fusion reaction at 37°C. After completion of the cell fusion reaction (for a total of 60 minutes at 37”), the fusion indexes (F.I.) were estimated.
serially diluted twofold with Hanks’ solution; samples of each dilution were mixed in test tubes with an equal volume of HVJ at a concentration of 16,000 or 8000 HAU/ ml. After incubation for 60 minutes at 37” and then overnight at 4”, the hemagglutinating, hemolytic, and cell fusion activities were determined. The results are shown in Fig. 2. The HA and HL activities of the control sample, not containing antiserum, showed the highest titers, and these activities decreased as the concentration of antiserum was increased. But the cell fusion reaction showed an abnormal behavior. Samples containing small doses of antiserum had higher cell fusion activities than the control sample. When 16,000 HAU of HVJ per milliliter were used, the highest fusion index was obtained in the sample containing antiserum diluted 2-*, and when 8000 HAU of HVJ per milliliter were used, the highest titer was obtained in the sample containing antiserum diluted 2-j. This indicates that a certain ratio of antiserum concentration to virus titer is required for enhancement of the cell fusion reaction. In Plate 1, a photograph of cells fused with HVJ alone is compared wit,h that of cells fused by a mixture of HVJ and anti-HV,J serum.
400
OKADA,
YAMADA,
AND TADOKORO HA 16384 4096 1024 256
6
Antiserum
dilution
factor
2. Anti-HVJ serum effect on HA, HL, and cell fusion activities of HVJ. An HVJ suspension of 16,000 (left-hand figure) or 8000 HAU/ml (right-hand figure) and a serially diluted anti-HVJ serum were mixed in equal volume. After incubation for 60 minutes at 37” and then overnight at 4”, HA, HL, and cell fusion activities of these samples were determined. O---O HAU/ml; A---A HL titer (optical density); 0-0 F.I. FIG.
When the cell fusion reaction was performed with a constant concentration of ETC and various titers of the virus, the degree of fusion increased as the virus titer was increased. However, t’here was a limit to the degree of fusion, and above this limit the degree of fusion decreased inversely with the virus titer. In Table 1, this limit was 5.5 F.I. in the sample containing 0.5 ml of the virus (20,480 HAU/ml). The decrease of the degree of cell fusion when virus titers above this value were used may be due to a kind of mutual interference of excess virus particles adsorbed onto ETC, or may be due to deterioration of the condition of the cells resulting from the adsorption of many virus particles. However, when virus samples containing 10,240 HAU/ml were mixed with antiserum of various concentrations, the degree of the cell fusion reaction exceeded the limit found in the absence of antiserum. As indicated in Table 1, the sample which showed a decrease of HA titer from the control value of 5120 to 640, had the highest degree of fusion (9.8 F.I.), and this value was not attained in the above case without antiserum. A purified y-globulin fraction of the antiHV.J serum behaved similarly to whole antiserum. Normal rabbit serum had no cell fusion enhancing or inhibiting activity, or HA-inhibiting activity (Table 2).
Differential Centrifugation of a Mixture HVJ and Anti-HVJ Serum
of
Two milliliters of a solution containing 8000 HAU/ml of HVJ were mixed with an equal volume of an anti-HVJ serum diluted to a concentration which gave maximal enhancement of the cell fusion reaction. After incubation at 37” for 60 minutes and then overnight at 4”, the sample was centrifuged at 1000 g for 20 minutes, the supernatant was removed, and the sediment was resuspended to the original volume in Hanks’ solution. One part of the supernatant was recentrifuged at 34,850 g for 60 minutes. The supernatant was removed and the sediment was resuspended to the original volume in Hanks’ solut,ion. Then, t,he HA titer and cell fusion activity of each fraction were determined. As a control, Hanks’ solution was added to an HVJ sample and treated by the same procedure. As indicated in Table 3, the HA and cell fusion activities of the control sample were sedimented by centrifugation only at 34,850 g. In the sample containing antiserum, the HA titer decreased from the cont’rol value of 4096 to 256 HAU, and the cell fusion activity increased
from
the
control
value
of
2.5
to 4.8 F.I. The HA and cell fusion activities sedimented together on low speed centrifugation
at 1,000 g. This indicated
t’hat most,
Normal IWC tLrid fused ETC caused by HVJ alone or a mixlure of HVJ :tnd an&HVJ S~IWII. “A” sl~ows normal at 37°C for 60 min; “IS” shows ETCIafter treatment with HVJ (4,096 ITAU/ml) for 60 min :It 37°C; “C” shows ETC in which the HVJ suspension (8,192 HAU/rnl) and diluted anti-HVJ strum were mixed in equ:tl volume.
PLATE I cont,rol ETC nfter incubation after treatment with a sample
402
OKADA, TABLE
CELL
YAMADA,
1
FUSION ACTIVITY OF HVJ AND THAT MIXED SAMPLES CONTAINING HVJ AND
OF
ANTI-HVJ SERUMS
HVJ
(HAU/ml)
Anti-HVJ serum (dilution factor)
Residual HA activity of mixed sample
-
-
40,960 20,480 10,240 5,120 2,560 1,280 640
-
10,240 10,240 10,240 10,240 10,240 10,240 10,240
20 2-l 2-2 2-3 2-4 2-5 2-e
F.I.
-
3.3 5.5 3.1 2.0 1.3 0.9 0.4
40 80 640 1280 2560 5120 5120
0.6 1.3 9.8 7.0 4.1 2.1 2.3
n In the experiment described in the upper half of this table, 0.5 ml of each HVJ sample was added to an equal volume of ETC and the fusion index was estimated after incubation for 60 minutes at 37”. In the experiment described in the lower half, 10,240 HAU/ml of HVJ was mixed with an equal volume of each dilution of antiserum, and after incubation at 37” for 60 minutes and then overnight at 4”, 0.5 ml of this mixture was added to ETC for determination of F.I.
TABLE
2
THE EFFECT OF NORMAL RABBIT SERUM ON THE CELL FUSION REACTION
Material added to HVJ sample
Serum dilution
HAU/ml
F.I.
20 2-l 2-z 2-3
4096 4096 4096 4096
2.9 3.3 3.2 3.3
-
4096
3.1
Normal rabbit serum
Hanks’ tion
solu-
0 An HVJ suspension (8192 HAU/ml) was mixed with normal or with diluted rabbit serum in equal volume.
AND TADOKORO
of the virus particles had combined with antibodies and the resulting large flocculates contained most of the HA and cell fusion activities of the sample. Efect of Heterologous Antisera on the Cell Fusion Reaction Antisera against other viruses of the myxovirus group were tested to see whether they had the same activity as anti-HVJ serum. One-milliliter aliquots of an anti-NDV serum, serially diluted twofold with Hanks’ solution, were mixed in test tubes with equal volumes of HVJ of a constant concentration of 16,000 HAU/ml. The mixtures were tested by the same procedure as was used for anti-HVJ serum. As indicated in Fig. 3, beginning with a dilution of antiNDV serum of less than 2-2 there was a decrease in HA activity. There also seemed to be an enhancement of the cell fusion reaction at 2-2 and 2-l dilutions of the antiserum. The antiserum used in the above experiment showed a titer of 1024 HIU against 4 HAU of NDV, but its HI titer against 4 HAU of HVJ was less than 32 units. However, when the sample containing concentrated HVJ was mixed with the antiNDV serum, heavy flocculation of the virus occurred and the HA titer decreased. This was because an antibody common to NDV and HVJ particles was present which differed from that corresponding to the HA activity. The decrease in the HA titer, shown in Fig. 3, may be due to virus flocculation resulting from an antibody against the common antigen, and not due to true neutralization of HA activity by a specific antibody. The appearance of enhancement of the cell fusion reaction in the sample showing a decrease in HA titer might indicate that agglutination or flocculation of the virus particles was correlated with this phenomenon. Actually, the flocculates of HVJ in the presence of the anti-NDV serum had a very high cell fusion activity as in the case of anti-HVJ serum. An anti-PR8 serum, showing a titer of 1024 HIU against PR8 virus and a titer of 32 HIU against HVJ, also showed these activities but they were rather weaker than
ANTISERUM
EFFECT
ON CELL FUSION REACTIOS
TABLE 3 DIFFERENTIAL CENTRIFUGATION OF A MIXTURE OF HVJ
AND ANTI-HVJ
F.I.
4096
2.5
Supernatant Precipitate
4096 512
2.5 0.3
Supernatant Precipitate
128 4096
0.0 2.4
256
4.8
Original” HVJ and Hanks’ solution
_---__-__
Centrifuged at 1000 g for 20 min --Centrifuged at 34,850 g for 60 min*
___Originalc
HVJ and antiserum
SERGAI
HAU/ml
Procedure
Sample
403
Centrifuged at 1000 g for 20 min
Supernatant Precipitate
32 256
0.5 5.9
Centrifuged at 34,850 g for 60 min*
Supernatant Precipitate
4 32
0.0 0.3
a Two milliliters of HVJ (8192 HAU/ml) was mixed with an equal volume of Hanks’ solution and incubated at 37” for 60 minutes and then overnight at 4”. b A part of the supernatant of material centrifuged at 1000 g was centrifuged at 34,850 g. c Two milliliters of HVJ (8192 HAU/ml) was mixed with an equal volume of a diluted anti-HVJ serum and incubated at 37” for 60 minutes and then overnight at 4”.
that of the anti-NDV serum. It is probable that HVJ, NDV, and PR8 virus particles contain normal antigens of the host cell [chorioallantoic membrane (CAM)], as indicated by Knight, who reported the existence of a host component in influenza virus particles of PR8 and Lee strains (1945). Thus, it can be presumed that (1) virus flocculation due to heterologous antiserum is initiated by the combination of a host component associated with the HVJ particle and the antibody against the host CAM component; (2) flocculation or agglutination of HVJ particles is essential for enhancement of the cell fusion reaction.
F. I
H.A. .
0.
H.A.
.-.-.-+-I
8192
6,
2048
512 .
F.I. .4*
128
Removal from Anti-NDV Serum of the Antibody against the Host Component The normal chorioallantoic fluid (CAF) of 30 eggs (13-day-old embryonated) was harvested and the supernatant, after centrifugation at 1000 g for 20 minutes, was ultracentrifuged at 105,500 g for 60 minutes. The sediment was suspended in 1.5 ml of antiNDV serum. The sample was kept at 37” for 60 minutes and then stored overnight at 4”. This sample was centrifuged at 105,500 g for 60 minutes, and the supernatant was
32 I
2-O
2-2 Antiserum
I control
2-4 dilution
factor
FIG. 3. Enhancement of the cell fusion reaction with anti-NDV serum. An HVJ suspension (16,000 HAU/ml) was mixed in equal volume with a serially diluted anti-NDV serum. After incubation for 60 minutes at 37” and then overnight at 4”, HA and cell fusion activities of these samples were determined.
404 HA
OKADA,
YAMAD.4,
F I
5120, 5
i.
5.. 320 2, 80. I
:10-
;. ‘\ 9 : ‘. ..’ J -A ti+--. ‘0 \.__A-.-$.~:Ac=:=Ao
c I-
FIG. 1. Comparison of ant,i-NDV serum :rbsorbed u-ith normal CAE’ components md nonabsorbed anti-NDV serum. Anti-NDV strum, absorbed twice with sedimentable components in normal CAF, or a nonabsorbed control, were mixed with an equal volume of HVJ (10,210 HAL/ ml). After incubation at 37” for 00 minutes and then overnight at 4”, HA and cell fusion activities and the degree of virus flocculation of these samples were determined. 0 - - - 0 HAU/ml, @---a F.T., and 0 . - . C protein content of the virus flocculates of the samples using nonab sorbed anti-XDV serum. a - ~- - L HAL/J~, A---A F.I., and A . - . A protein content of the virus flocculates of the samples using antiNDV serum absorbed with CAF components.
harvest.ed. This procedure of absorption with CAF components was repeated twice. As a control, antiserum alone was ccntrifuged at 105,500 g. One-milliliter aliquots of these antiseruln samples, serially diluted twofold with Hanks’ solution, were mixed in test, tubes with an equal volume of HV,J of a constant concentration of 10,240 HAU/ml. The mixtures were incubated at 37” for 1 hour and then overnight at 4”. Then, the HA and cell fusion activities of these samples were dctermined. As shown in Fig. 4, in the expcrimental series in which control anti-NDV serum was used, the HA titer decreased as the degree of virus flocculation incrcsscd. Enhancement of the cell fusion reaction was observed in some samples. On the contrary, when antiserum absorbed twice with normal CAF components was used, flocculation of
,IXD
TADOKORO
HVJ particles was not apparent, the HA titer did not decrease and the dcgrce of the cell fusion reaction was constant and indcpendent of the amount of serum added. This result clearly indicates t,hat most of the antibody forming a complex with HVJ particles is antibody against a normal component, and that this virus-antibody complex had a high cell fusion activity. MoreOTW. wc have found that, an antiserum agsinst normal chorioallantoic membrane was also capable of enhancing the cell fusion reartion and dccrcasing the HA titer of the virus.
In the sample containing a largcx dose of anti-NDV serum, the cell fusion activity, and the HA activity becrur~ very low, as shown in Figs. 3 and 4. Tl1is might, be due to the fact, that virus flocculatcts grew into very large clumps and w-cnre far fewer in number &an the ETC used for the cell fusion reaction or than red blood cells used for HA titration. Hemozd
of the .4ntiDody against the Host
To st,udy the role of the antibody against the host component in anti-HV,J serum, a 2-1111aliquot of anti-IIV,J serum was ab~orbcd twice with normal CAF cornponcnts l1rcparcd from 50 embryonated eggs. It w::s found that the cell fusion enhancing activity, the HA neutralizing activity: and tlrc HVJ flocculating activity of the antiserum rcmaincd essentially the same as that of nontreated control antiserum, as indicated in Fig. 5. Immunological tests were then made to dctcrmine whether the antibody against the host component in the antiserum was rcmoved by the absorption procedure. A gel diffusion method was employed, using antigens prepared by Hosaka’s method (Hosaka et al., 190). Concentrated HVJ was disintegrated with ether in the presence of Emasol (a nonionizing detergent like Tween 80) and centrifuged at 2000 rpm for 5 minutes; the water phase was separated from the ether phase and used as the HV,J antigen. As normai host antigen, the sedimentable components tlicnterl
in normal CAF were used. As inin Plate 2, six precipitation lines
ASTISERUM
EFFECT
ON CELL
appeared between the HVJ antigen and the control antiserum. These lines were arbitrarily numbered as 1, l’, 2, 5, J$, and 5, respectively, in order from the one nearest to the HVJ antigen. Lines nos. 2 and 5 did not appear between the HVJ antigen and the reabsorbed antiserum. Two lines appeared between the normal CAF component and the control antiserum, but not between the normal component and the reabsorbed antiserum. One line appeared between the control serum and the reabsorbed serum, and t’his seemed to be the same as line no. 2 and a line between the normal component and the control serum. This indicates that the antigen corresponding to line no. 2 was common to HVJ and the normal host component, that the antibody had been completely removed from the reabsorbed antiserum, and that a remnant of the added antigen remained in the serum. The other line between the normal component and the control antiserum may correspond to line no. 5, but this did not appear between HVJ ant.igen and the reabsorbed antiserum or the normal component and the reabsorbed antiserum. It is therefore concluded that antibodies against the normal components in the ant,i-HVJ serum were removed by reabsorption w&h normal CAF components. When t’he anti-HVJ serum was absorbed with KDV particles prepared from infected CAF, the cell fusion enhancing, the HA and the HVJ flocculating neutralizing, actirit,ies of the antiserum did not change. Only when the antiserum was absorbed with HVJ particles, did all these activities disappear, as indicated in Fig. 5. From these findings it is concluded that the antibodies against the normal host components in the anti-HVJ serum had scarcely any significance in the decrease of HA titer, flocculation of the virus, or enhancement of t,he cell fusion activity of the virus. It is the antibodies against the specific HVJ antigens that play a prominent role in these nctivit,ies. Comparison of the Cell Agglutination Activity and the Cell Fusion Activity of HVJ with Antiserum Since the cells are in suspension in the ETC-HVJ system, the first step in cell
FUSION
REACTION
405
HA 5120.. ,. FI. 00 ,’
1260
320,.
6,
4’
80.
2,
20-
o-
FIG. 5. Effects of anti-HVJ serum absorbed with normal CAF components, or with HVJ particles, on HVJ. Anti-HVJ serum, absorbed with sedimentable components in normal CAF, or with purified HVJ particles, or a nonabsorbed control, were mixed with an equal volume of HVJ (10,240 HAU/ml) After incubation at 3” for 60 minutes and then overnight at -1”, the HA and cell fusion activities and the degree of virm flocculation of these samples Here determined. 0 - - - 0 HAU/ ml, @---a F.I., and (> - (> protein content of the virus flocculates of the samples using nonabsorbed anti-HVJ serum. n - - - n HAU/ml, A--A F.I., and A . - A protein content of the virus flocculates of the samples using antiserum absorbed with CAF components. V - - - V HAL/ml, v---‘I F.I., and V . - V protein content of the virus flocculates of the samples using antiserum absorbed with HVJ particles.
fusion is contact between participant cells. This step is caused by the HA activity of HVJ. When ETC and HVJ were mixed, the virus particles were promptly adsorbed onto the surface of the cells and then these cells agglutinated together. The degree of cell fusion after completion of the reaction at 37” may be influenced by the extent of clumping of the cells produced by HVJ. This clumping increases with the number of the virus particles added to ETC. The succeeding steps in the fusion reaction occurred when the cell clump was incubated at 37”, some cells in the clump fused together and the others became dispersed, liberating free cells into the medium. The factor affecting the reaction in these latter
PLATE 2 analysis of anti-HVJ serum absorbed with normal CAF components by the gel diffusion mc(hwl. .41, HVJ an&n (disintegrated with ether); A2, normal C-IF components; El, anti-HVJ serum (unabsorbed); B2, anti-HVJ serum (absorbed with sedimentable components in normal CAF).
al-7
ANTISERUM
EFFECT
ON CELL FUSION REACTION
steps is the cell fusion activity of t,he virus adsorbed onto the cells, unlike the HA activity (Okada and Tadokoro, 1962). A study was made of whether the enhancement of the cell fusion reaction on addition of antiserum is due to an increase in the degree of cell agglutination or to a reinforcement of the cell fusion activity of HVJ. Aliquots of 1 ml of anti-HVJ serum, diluted serially twofold with Hanks’ solution, and an equal volume of HVJ containing 10,240 HAU/ml were mixed and kept ::t 37” for 60 minutes and then overnight at, 4”. Then, 0.5 ml of the mixture of virus and antiserum and an equal volume of ETC suspension containing 1.2 X 10’ cells/ml A.I.
F. I.
407
were mixed in two series of tests tubes at 4”. After standing for 15 minutes at 4”, the tubes of the one series were gently agitated and the samples were introduced into a Fuchs-Rosenthal cytometer and photographed at low magnification. The total number of free cells and cell clumps in the samples were counted from the photographs. The cell agglutination index (A.I.) was calculated as; A.I. =
cell number of nonvirus control tube -1 total number of free cells plus cell clumps in test tube
The other series of tubes was incubated for 60 minutes with shaking at 37” and then their fusion indexes mere determined. The results are shown in Fig. 6. The cell agglutination activity was highest in the control virus sample without antiserum and decreased as the concentration of the antiserum was increased. On the contrary, the cell fusion activity increased in some samples containing a low dose of antiserum. This indicates that the enhancement of the cell fusion reaction is due to an increase of the cell fusion activity of HVJ by the addition of the antiserum, and is not due to an increase of the degree of cell agglutination. DISCUSSION
Antiserum
dilution
factor
FIG. 6. Comparison of cell agglutination activity and cell fusion activity of mixtures of HVJ and anti-HVJ serum. An HVJ suspension (10,240 HAU/ ml) and a serially diluted anti-HVJ serum were mixed in equal volume and incubated for 60 minutes at 37” and then overnight at 4”. One-half milliliter of the mixtures and an equal volume of ETC suspension were then mixed in two series of test tubes. The degree of cell agglutination (A.I.) at 4” was determined from the one series of tubes, and the degree of cell fusion (F.I.) after incubation at 37” for 60 minutes was estimated from the other series of tubes. A - -- A cell agglutination index at 4”; O-O cell fusion index after incubation at 37”.
From the results of previous investigations (Okada, 1962a; Okada and Tadokoro, 1962), it may be concluded that an active substance for the cell fusion reaction is present on the surface of the HVJ particle, and the cell fusion reaction is initiated by a direct reaction of this active substance on the virus with the surface of the participant cells, not by virus infection. The cell fusion reaction in the ETC-HVJ system may progress as follows. The first step is the adsorption of virus particles onto the cells, followed by the agglutination of the cells. The second step is a kind of loosening or disconnecting of the surface structure of the participant cells at 37”. The third step is a connection of the loosened parts of neighboring cell surfaces. If there is no neighboring cell, the loosened site on the cell surface may reconnect with the
408
OKADA,
YAMADA,
main part of the cell. The fourth step is a mixing of the cytoplasm of the participant cells and a formation of a spherical polynuclear cell. The first step in this series of reactions is caused by the HA activity of the virus at 4”. The cell fusion activity of the virus may be involved in the second and third steps. The effect of a large titer of anti-HVJ serum during the cell fusion reaction suggests that at 37” the second and third steps arc complete within 5 minutes from the beginning of the fusion reaction. The fourth step seems to be independent of the activities of the virus. A characteristic of the cell fusion reaction is the necessity for adsorption of many virus particles onto one cell. Thus 1300 virus particles must be adsorbed onto one cell for a 50% decrease in cell number due to fusion of cells (F.I. = 1.0) under good experimental conditions and the degree of the cell fusion reaction increases with the number of adsorbed virus particles (Okada and Tadokoro, 1962). The correlation of the number of adsorbed virus particles with t.he degree of cell fusion may be replaced by a relationship between the density of virus particles per unit of cell surface and the degree of cell fusion. Moreover, only virus particles present between parts of two or more cells which are in contact may participate in the actual cell fusion reaction, and the density of the virus (or the active cell fusion substance of the virus) at this position may be correlated with the efficiency of the change from free to fused cells (or the completion of the second and third steps of the cell fusion reaction). When anti-NDV serum’ was used, the antigen-antibody complex only formed between antigen molecules of host components associated with HVJ particles and antibodies against their antigens, and the complex had a high fusion activity. In this case, the cell fusion activity of the virus in the flocculate may not be neutralized. When virus particles aggregated by anti-NDV serum are adsorbed onto the cells, the adsorption of these aggregates by the free cells may occur. In this case, the density of the active cell fusion substance of the virus aggregate at the part in contact with the cell is higher than when the same
AND TADOKORO
number of free virus particles are adsorbed at random onto the cell surfaces when no anti-NDV serum is present. This phenomenon may be one of the chief reasons why the cell fusion reaction is enhanced by the addition of anti-NDV serum. The increase in cell fusion on addition of anti-HVJ serum, may be due to the same phenomenon, although the activity was not removed by absorption with normal CAF components. If the relative amount of antibodies against virus specific antigens is very much greater in anti-HVJ serum than the amount of antibodies against normal host components, the total activity corresponding to flocculation of HVJ in the serum may be affected very little by removal of antibodies against host components. Moreover, if antibody against the substance causing cell fusion activity is present in small amounts in anti-HVJ serum, the virus flocculation may be produced by combination of antigens and antibodies other than those corresponding to the cell fusion activity. Thus, in this case, the cell fusion activity of the virus in the flocculate might not be neutralized, as in the case with heterologous antiserum, under conditions when the cell fusion reaction was enhanced. If so, the substance showing cell fusion activity in the virus particle may consist of components with a low antibody response. In the HVJ and anti-HVJ serum system, neutralization of the cell lytic activity (HL activity) of the virus with the antiserum may also play a role in the phenomenon of enhancement. Other factors involved in the enhancement phenomenon, such as the influence of the antiserum itself, must also be considered. Normal rabbit serum did not have any affect on the reaction. Ishizaka et al. (1962) indicated that antibody molecuIes, brought into apposition by antigen, interact with each other. The antibody antigen interaction and/or consequent changes in the antibody molecules might be responsible for various biological phenomena. In our experiments, the antibodies which formed a complex with HVJ particles would also acquire
a new biological
activity,
as shown
by Ishizaka et al. To determine whether antibody molecules which are altered by
ANTISERUM
EFFECT
ON CELL
combination with HVJ particles play a role in the cell fusion reaction, the cell fusion reaction was measured using the NDV and anti-NDV complex, because our NDV strain had cell agglutination but not cell fusion activity (Okada and Tadokoro, 1962). The NDV and anti-NDV serum complex, like NDV alone, had no cell fusion activity. In another experiment, a distribution of cell strains or cells showing cell fusion by HVJ was determined and a tendency was observed for undifferentiated cells, but not differentiated celhs, to have a high cell fusion capacity (Okada and Tadokoro, 1963). In the experimental series, leukemia cells from a pat,ient with myeloic leukemia were fused by HVJ, whereas mature polymorpholeucocytes from a patient with rheumatic fever were not. The efficiency of the cell fusion reaction with leukemia cells was enhanced with anti-HV.J serum as in the case of ETC, but the polymorpholeucocytes did not fuse on addition of a mixture of HVd and antiserum, as with HVJ alone. ACKNOWLEDGMENTS The authors wish to express their thanks to Dr K. Fukai, Dr. T. Amano, Dr. M. Yoneda, and Dr. Y. Hosaka of this Institute for their advice. Thanks are also expressed to Dr. M. Shimoyama and Miss F. Murayama for technical assistance. REFERENCES COWIE, D. B., SPIEGELMAN, S., ROBERTS, R., and DUERKSEN, J. D. (1961). Ribosome-bound pgalactosidase. Proc. Natl. Acad. Sci. U.S. 47, 114122. GRANOFF, A., and HENLE, W. (1954). Studies on the hemolytic activity of Newcastle disease virus.
J. Immunol. 72,322-328. HOSAKA, Y., HOSOKAWA, Y., and FUKAI, K. (1960).
FUSION
REACTION
409
Structure of HVJ. I. Two kinds of subunits of HVJ. Bike,l’s J. 3, 27-40. ISHIZAKA, Ii., 1~~1z.4ri.4, T., and SUGAHARA, T. (1962). Biological activity of soluble antigenantibody complexes. VII. Role of an antibody fragment in the induction of biological :tctivities. .I. Itnm unol. 88, 690-701. KSIGTIT, C. A. (1945). Precipitin reactions of highly purifit~d influenza virusIs and related matrrinls. J. Exptl. Med. 82,281-294. Low-su, 0. H., ROSEBROXH, N. J., FARR. L., and R.~XDALL, R. J. (1951). Protein measurement with the Folin phenol reagent, .I. BioZ. C’hc,~l. 193, 265-275. OK;ADA, Y. (1958). The fusion of Ehrlich’s tumor cells caused by HV-J virus itl vifTo. Bikcn’s J. 1, 103-110. OKADA, Y. (1962n). Analysis of giant polgnuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. I. Microscopic observation of giant polynuclrar cell formation. Ex7jtl. Cell Res. 26,98-107. OKAD.~, Y. (196213). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. III. Relationship between cell condition nntl fusion reaction or cell degeneration reaction. Ezptl. Cell Res. 26, 119128. OK.~D.~, T., and TADOICORO,J. (1962). .~nnlysis of giant polynuclrar cell formation caused by HVJ virus from Ehrlich‘s ascitcs tumor cells. II. Quantitative analysis of giant polynuclr::rr cell formation. Exptl. Cell R(a.s. 26, 108-118. OKADA, Y., and T.~DOIWRO, J. (1963). The distribution of cell fusion capacity among several cell strains or cells caused by HVJ. Ez(jf/. (‘c2l 1L’e.s. 32(3!, the December issue. OKADA, Y., SWXJKI, T., and HOSAKA, T. (1957). Interaction between influenza virus and Ehrlich’s tumor cells. III. Fusion phenomenon of Ehrlich’s tumor cells by the action of HVJ, Z strain. Med. J. Osaka Univ. 7,709-717. OKADA, T., NISHIDA, S., and TADOBORO, J. (1961). Correlation between the hemagglutination titer and the virus particle number of HVJ. B&en’s J. 4, 209-214.
VIROLOGY
Effect
(1964)
of Antiserum YOSHIO
Department
OKADA, of Preventive
on the Cell KAZUKO
Fusion
YAMADA,
Reaction AND
October
by HVJ’
JUN T,4DOKOR02
Medicine, The Research Institute Osaka University, Osaka, Japan Accepted
Caused
for Microbial
Diseases,
15, 1963
The cell fusion reaction of Ehrlich ascites tumor cells caused by HVJ was completely inhibited by using a large amount. of anti-HVJ serum. However, when diluted antiserum was added to the HVJ sample, the cell fusion reaction was enhanced. In the sample, showing maximal enhancement of the reaction, almost all the cell fusion activity was found in virus flocculates which were sedimentable by low speed centrifugation. When antiserum against other myxoviruses, e.g., NDV or PRS, was mixed with the HVJ sample, virus flocculation also occurred together with enhancement of the cell fusion reaction. Both activities, the HVJ flocculating and the cell fusion-enhancing activities, were lost after absorption with sedimentable components in normal chorioallantoic fluid. The enhancement of the cell fusion reaction with anti-HVJ or antisera against other myxoviruses may be correlated with the formation of virus clumps, in which the cell fusion activity of the participant virus is kept as high as that of the free virus. INTRODUCTION
In 1957, Okada et al. reported the appearance of many giant polynuclear tumor cells within an hour after a large dose of HVJ had been inoculated into the abdominal cavity of a mouse implanted with Ehrlich ascites tumor cells; they showed that the cells agglutinated as a result of virus adsorption and fused together, and that multinucleated giant cells appeared. Subsequently this phenomenon was analyzed in vitro (Okada, 1958, 1962a,b; Okada and Tadokoro, 1962). As a continuation of this work, the effect of anti-HVJ serum on the cell fusion reaction was studied; the results are described in this report. The reaction was completely inhibited by the addition of a sufficiently large titer of the antiserum. But surprisingly, when a small dose of antiserum was added to the HVJ sample, the cell fusion 1 Hemagglutinating virus of Japan, Sendai virus. * Present address : Department of Osaka National Hospital.
synonym
:
Pediatrics, 397
reaction was strikingly enhanced compared with the effect of the virus only. Cowie et al. (1961) reported that exposure of ribosome-bound p-galactosidase to antiserum against P-galactosidase resulted in a three- to sixfold increase in activity. In this report, the enhancement of the cell fusion reaction by virus that has combined with a small amount of antibody is described and discussed. The inhibition caused by a large dose of the antiserum is also reported. MATERIALS
AND
METHODS
I’irus. The egg-adapted Z strain of HVJ was used. The virus was propagated in the allantoic sacs of lo-day-old chick embryos, using a diluted inoculum. The allantoic fluid was centrifuged at 1400 g for 20 minutes. Then the supernatant was centrifuged at 34,850 g for 30 minutes. The sediment was suspended in modified Hanks’ solution and was recent,rifuged at 1400 g for 20 minutes to eliminate aggregates. Cells. Ehrlich hypertetraploid ascites
398
OKADA,
YAMADA,
tumor cells (ETC) were withdrawn from the peritoneal cavity of adult Swiss ddO mice inoculated 7 days previously with 0.1 ml of tenfold diluted tumor-bearing ascites fluid. The cells were washed three or four times with modified Hanks’ solution to remove contaminating red cells and leucocytes and were suspended in the same solution at a concentration of 1.2 X IO7 cells/ml. Anti-HVJ, NDV, and PR8 sera. Aliquots of 1 ml of purified HVJ, Newcastle disease virus (NDV) or PR8 strain of influenza virus (10,000 hemagglutinating units per milliliter), propagated in embryonated eggs, were injected into a rabbit, alternately intravenously and intramuscularly once a week. One week after the third injection, the blood was withdrawn by cardiac puncture and the antisera were incubated at 56°C for 60 minutes. These antisera were supplied by Dr. Y. Hosaka of our laboratory. Also antisera obtained by immunization with mixed samples of viruses and incomplete adjuvant (composed of Arlacel and Drackeol No. 6) were used. Modified Hanks’ solution. Hanks’ balanced salt, solution, without glucose or bicarbonate, was used throughout the work. The pH was adjusted to 7.6 with tris(hybuffer droxymethyl) aminomethane-HCl (0.01 M). Hemagglutination (HA) titration. Salk’s pattern method employing 0.5 ml of 0.5% fowl red blood cells was used with phosphate-buffered saline as the diluent. Under these conditions, one HA unit corresponds to about 2.4 x lo7 virus particles (Okada et al., 1961). Determination
of hemolytic
(HL)
actiu-
One milliliter of the virus sample was mixed with 2 ml of 2% fowl red blood cell suspension. The mixture was kept in the cold for 15 minutes and then washed once with cold phosphate-buffered saline (pH 7.2)) and PBS was added to the original volume. The sample was put in a water bath at 37” for 60 minutes and agitated every 15 minutes. After incubation, the tube was centrifuged at 350 g for 5 minutes. The supernatant fluid was transferred to a calorimeter cuvette and the amount of hemoglobin liberated was determined specity.
AND
TADOKORO
trophotometrically at 540 mp. As a reference PBS was used in place of the virus. When a high concentration of HVJ was used, hemolysis was inhibited as reported by Granoff and Henle (1954) in a NDV-fowl red blood cell system. But if freeze-thawed virus was used, there was no inhibition and the degree of hemolysis increased with the amount of virus added. Moreover, the freeze-thawed virus behaved the same way with anti-HVJ serum as the native virus. For these reasons, only freeze-thawed (2 cycles) virus samples were used when HL activity was to be measured in the experiment. Cell fusion reaction. A 0.5-ml aliquot of the ETC sample containing 1.2 x IO7 cells/ml and an equal volume of virus sample were mixed in a test, tube. The tube was put in an ice bath for 15 minutes to allow cell agglutination. Then the tube was incubated in a water bath at 37” with shaking for 60 minutes. During the incubation the cells in the clumps fused together or became dispersed in the medium ; no cell clumps remained at the end of the incubation. In other words, the decrease in the cell number observed at the end of the reaction corresponded to the degree of cell fusion. After incubation the sample was cooled and mixed by pipette; an aliquot was introduced into a Fuchs-Rosenthal cytometer and photographed with a phase contrast, microscope at low magnification. The cell number of the sample was counted from ten negative plates (under these conditions, about 200 cells were photographed per plate of nonvirus control sample) and the “fusion index” was calculated according to the following formula (Okada and Tadokoro, 1962) : fusion index (F.1.) =
cell number
in nonvirus
cell number
control
in test tube
tube
-
1.0
Determination of the degree of flocculation of the virus with antiserum. A mixture
of virus suspension and trifuged at 1000 g for keeping the sample for and overnight at 4°C.
antiserum was cen20 minutes after 60 minutes at 37” The sediment was
ANTISERUM
EFFECT
389
ON CELL FUSION REACTION
washed with saline, and the protein content was estimated by the method of Lowry et nl. (1951). Agar gel difusion. The double diffusion technique of Ouchterlony was employed. RESULTS
Inhibition of the Cell Fusion Reaction with a Sufficient Titer of Anti-HVJ Serum Aliquots of 0.5 ml of a solution containing 8000 HAU of HVJ per milliliter were mixed in test tubes with an equal volume of a solution containing 1.2 x lo7 cells of ETC per milliliter and kept for 15 minutes at 4”. One milliliter of anti-HVJ serum (with a hemagglutination inhibition titer of 1024 units/ml against 4 HAU of HVJ) was introduced into one of the test tubes during this incubation at 4”, and the tube was incubated at 37” for 60 minutes with shaking. The other test tubes, after being kept at 4” for 15 minutes, were incubated at 37” with shaking, and the same amount of the antiserum was added during the course of the cell fusion reaction at 37”. After completion of the cell fusion reaction (for a total of 60 minutes at 37”), the cell fusion index (F.I.) of these samples was estimated. As indicated in Fig. 1, the cell fusion reaction was completely inhibited only when antiserum was added before incubation at 37” and the antiserum did not affect the reaction in the sample after incubation for 5 minutes or longer at 37”. This experiment showed that the initiation reaction of the cell fusion process with the virus was completed within 5 minutes at 37”. This is compatible with the results of previous studies on the mechanism of cell fusion obtained by electron microscopy, in which it was shown that the virus particles disappeared from the cell surface within 5 minutes on incubation at 37”, and at that time the cell membranes of neighboring cells had already fused together (Okada, 1962a). Enhancement of the Cell Fusion Reaction by a Small Amount of Anti-HVJ Serum One milliliter of anti-HVJ serum, with a titer of 1024 HIU (hemagglutination inhibition units) against 4 HAU of HVJ, was
A-!
0 5 IO
60 min
30
Time of antiserum
addition
FIG. 1. Inhibition of the cell fusion reaction with anti-HVJ serum. Aliquots of 0.5 ml of 8000 HAU of HVJ per milliliter were mixed with an equal volume of 1.2 X 10’ cells of ETC per milliliter in test tubes. One milliliter of undiluted anti-HVJ serum was introduced into the tubes during the cell fusion reaction at 37°C. After completion of the cell fusion reaction (for a total of 60 minutes at 37”), the fusion indexes (F.I.) were estimated.
serially diluted twofold with Hanks’ solution; samples of each dilution were mixed in test tubes with an equal volume of HVJ at a concentration of 16,000 or 8000 HAU/ ml. After incubation for 60 minutes at 37” and then overnight at 4”, the hemagglutinating, hemolytic, and cell fusion activities were determined. The results are shown in Fig. 2. The HA and HL activities of the control sample, not containing antiserum, showed the highest titers, and these activities decreased as the concentration of antiserum was increased. But the cell fusion reaction showed an abnormal behavior. Samples containing small doses of antiserum had higher cell fusion activities than the control sample. When 16,000 HAU of HVJ per milliliter were used, the highest fusion index was obtained in the sample containing antiserum diluted 2-*, and when 8000 HAU of HVJ per milliliter were used, the highest titer was obtained in the sample containing antiserum diluted 2-j. This indicates that a certain ratio of antiserum concentration to virus titer is required for enhancement of the cell fusion reaction. In Plate 1, a photograph of cells fused with HVJ alone is compared wit,h that of cells fused by a mixture of HVJ and anti-HV,J serum.
400
OKADA,
YAMADA,
AND TADOKORO HA 16384 4096 1024 256
6
Antiserum
dilution
factor
2. Anti-HVJ serum effect on HA, HL, and cell fusion activities of HVJ. An HVJ suspension of 16,000 (left-hand figure) or 8000 HAU/ml (right-hand figure) and a serially diluted anti-HVJ serum were mixed in equal volume. After incubation for 60 minutes at 37” and then overnight at 4”, HA, HL, and cell fusion activities of these samples were determined. O---O HAU/ml; A---A HL titer (optical density); 0-0 F.I. FIG.
When the cell fusion reaction was performed with a constant concentration of ETC and various titers of the virus, the degree of fusion increased as the virus titer was increased. However, t’here was a limit to the degree of fusion, and above this limit the degree of fusion decreased inversely with the virus titer. In Table 1, this limit was 5.5 F.I. in the sample containing 0.5 ml of the virus (20,480 HAU/ml). The decrease of the degree of cell fusion when virus titers above this value were used may be due to a kind of mutual interference of excess virus particles adsorbed onto ETC, or may be due to deterioration of the condition of the cells resulting from the adsorption of many virus particles. However, when virus samples containing 10,240 HAU/ml were mixed with antiserum of various concentrations, the degree of the cell fusion reaction exceeded the limit found in the absence of antiserum. As indicated in Table 1, the sample which showed a decrease of HA titer from the control value of 5120 to 640, had the highest degree of fusion (9.8 F.I.), and this value was not attained in the above case without antiserum. A purified y-globulin fraction of the antiHV.J serum behaved similarly to whole antiserum. Normal rabbit serum had no cell fusion enhancing or inhibiting activity, or HA-inhibiting activity (Table 2).
Differential Centrifugation of a Mixture HVJ and Anti-HVJ Serum
of
Two milliliters of a solution containing 8000 HAU/ml of HVJ were mixed with an equal volume of an anti-HVJ serum diluted to a concentration which gave maximal enhancement of the cell fusion reaction. After incubation at 37” for 60 minutes and then overnight at 4”, the sample was centrifuged at 1000 g for 20 minutes, the supernatant was removed, and the sediment was resuspended to the original volume in Hanks’ solution. One part of the supernatant was recentrifuged at 34,850 g for 60 minutes. The supernatant was removed and the sediment was resuspended to the original volume in Hanks’ solut,ion. Then, t,he HA titer and cell fusion activity of each fraction were determined. As a control, Hanks’ solution was added to an HVJ sample and treated by the same procedure. As indicated in Table 3, the HA and cell fusion activities of the control sample were sedimented by centrifugation only at 34,850 g. In the sample containing antiserum, the HA titer decreased from the cont’rol value of 4096 to 256 HAU, and the cell fusion activity increased
from
the
control
value
of
2.5
to 4.8 F.I. The HA and cell fusion activities sedimented together on low speed centrifugation
at 1,000 g. This indicated
t’hat most,
Normal IWC tLrid fused ETC caused by HVJ alone or a mixlure of HVJ :tnd an&HVJ S~IWII. “A” sl~ows normal at 37°C for 60 min; “IS” shows ETCIafter treatment with HVJ (4,096 ITAU/ml) for 60 min :It 37°C; “C” shows ETC in which the HVJ suspension (8,192 HAU/rnl) and diluted anti-HVJ strum were mixed in equ:tl volume.
PLATE I cont,rol ETC nfter incubation after treatment with a sample
402
OKADA, TABLE
CELL
YAMADA,
1
FUSION ACTIVITY OF HVJ AND THAT MIXED SAMPLES CONTAINING HVJ AND
OF
ANTI-HVJ SERUMS
HVJ
(HAU/ml)
Anti-HVJ serum (dilution factor)
Residual HA activity of mixed sample
-
-
40,960 20,480 10,240 5,120 2,560 1,280 640
-
10,240 10,240 10,240 10,240 10,240 10,240 10,240
20 2-l 2-2 2-3 2-4 2-5 2-e
F.I.
-
3.3 5.5 3.1 2.0 1.3 0.9 0.4
40 80 640 1280 2560 5120 5120
0.6 1.3 9.8 7.0 4.1 2.1 2.3
n In the experiment described in the upper half of this table, 0.5 ml of each HVJ sample was added to an equal volume of ETC and the fusion index was estimated after incubation for 60 minutes at 37”. In the experiment described in the lower half, 10,240 HAU/ml of HVJ was mixed with an equal volume of each dilution of antiserum, and after incubation at 37” for 60 minutes and then overnight at 4”, 0.5 ml of this mixture was added to ETC for determination of F.I.
TABLE
2
THE EFFECT OF NORMAL RABBIT SERUM ON THE CELL FUSION REACTION
Material added to HVJ sample
Serum dilution
HAU/ml
F.I.
20 2-l 2-z 2-3
4096 4096 4096 4096
2.9 3.3 3.2 3.3
-
4096
3.1
Normal rabbit serum
Hanks’ tion
solu-
0 An HVJ suspension (8192 HAU/ml) was mixed with normal or with diluted rabbit serum in equal volume.
AND TADOKORO
of the virus particles had combined with antibodies and the resulting large flocculates contained most of the HA and cell fusion activities of the sample. Efect of Heterologous Antisera on the Cell Fusion Reaction Antisera against other viruses of the myxovirus group were tested to see whether they had the same activity as anti-HVJ serum. One-milliliter aliquots of an anti-NDV serum, serially diluted twofold with Hanks’ solution, were mixed in test tubes with equal volumes of HVJ of a constant concentration of 16,000 HAU/ml. The mixtures were tested by the same procedure as was used for anti-HVJ serum. As indicated in Fig. 3, beginning with a dilution of antiNDV serum of less than 2-2 there was a decrease in HA activity. There also seemed to be an enhancement of the cell fusion reaction at 2-2 and 2-l dilutions of the antiserum. The antiserum used in the above experiment showed a titer of 1024 HIU against 4 HAU of NDV, but its HI titer against 4 HAU of HVJ was less than 32 units. However, when the sample containing concentrated HVJ was mixed with the antiNDV serum, heavy flocculation of the virus occurred and the HA titer decreased. This was because an antibody common to NDV and HVJ particles was present which differed from that corresponding to the HA activity. The decrease in the HA titer, shown in Fig. 3, may be due to virus flocculation resulting from an antibody against the common antigen, and not due to true neutralization of HA activity by a specific antibody. The appearance of enhancement of the cell fusion reaction in the sample showing a decrease in HA titer might indicate that agglutination or flocculation of the virus particles was correlated with this phenomenon. Actually, the flocculates of HVJ in the presence of the anti-NDV serum had a very high cell fusion activity as in the case of anti-HVJ serum. An anti-PR8 serum, showing a titer of 1024 HIU against PR8 virus and a titer of 32 HIU against HVJ, also showed these activities but they were rather weaker than
ANTISERUM
EFFECT
ON CELL FUSION REACTIOS
TABLE 3 DIFFERENTIAL CENTRIFUGATION OF A MIXTURE OF HVJ
AND ANTI-HVJ
F.I.
4096
2.5
Supernatant Precipitate
4096 512
2.5 0.3
Supernatant Precipitate
128 4096
0.0 2.4
256
4.8
Original” HVJ and Hanks’ solution
_---__-__
Centrifuged at 1000 g for 20 min --Centrifuged at 34,850 g for 60 min*
___Originalc
HVJ and antiserum
SERGAI
HAU/ml
Procedure
Sample
403
Centrifuged at 1000 g for 20 min
Supernatant Precipitate
32 256
0.5 5.9
Centrifuged at 34,850 g for 60 min*
Supernatant Precipitate
4 32
0.0 0.3
a Two milliliters of HVJ (8192 HAU/ml) was mixed with an equal volume of Hanks’ solution and incubated at 37” for 60 minutes and then overnight at 4”. b A part of the supernatant of material centrifuged at 1000 g was centrifuged at 34,850 g. c Two milliliters of HVJ (8192 HAU/ml) was mixed with an equal volume of a diluted anti-HVJ serum and incubated at 37” for 60 minutes and then overnight at 4”.
that of the anti-NDV serum. It is probable that HVJ, NDV, and PR8 virus particles contain normal antigens of the host cell [chorioallantoic membrane (CAM)], as indicated by Knight, who reported the existence of a host component in influenza virus particles of PR8 and Lee strains (1945). Thus, it can be presumed that (1) virus flocculation due to heterologous antiserum is initiated by the combination of a host component associated with the HVJ particle and the antibody against the host CAM component; (2) flocculation or agglutination of HVJ particles is essential for enhancement of the cell fusion reaction.
F. I
H.A. .
0.
H.A.
.-.-.-+-I
8192
6,
2048
512 .
F.I. .4*
128
Removal from Anti-NDV Serum of the Antibody against the Host Component The normal chorioallantoic fluid (CAF) of 30 eggs (13-day-old embryonated) was harvested and the supernatant, after centrifugation at 1000 g for 20 minutes, was ultracentrifuged at 105,500 g for 60 minutes. The sediment was suspended in 1.5 ml of antiNDV serum. The sample was kept at 37” for 60 minutes and then stored overnight at 4”. This sample was centrifuged at 105,500 g for 60 minutes, and the supernatant was
32 I
2-O
2-2 Antiserum
I control
2-4 dilution
factor
FIG. 3. Enhancement of the cell fusion reaction with anti-NDV serum. An HVJ suspension (16,000 HAU/ml) was mixed in equal volume with a serially diluted anti-NDV serum. After incubation for 60 minutes at 37” and then overnight at 4”, HA and cell fusion activities of these samples were determined.
404 HA
OKADA,
YAMAD.4,
F I
5120, 5
i.
5.. 320 2, 80. I
:10-
;. ‘\ 9 : ‘. ..’ J -A ti+--. ‘0 \.__A-.-$.~:Ac=:=Ao
c I-
FIG. 1. Comparison of ant,i-NDV serum :rbsorbed u-ith normal CAE’ components md nonabsorbed anti-NDV serum. Anti-NDV strum, absorbed twice with sedimentable components in normal CAF, or a nonabsorbed control, were mixed with an equal volume of HVJ (10,210 HAL/ ml). After incubation at 37” for 00 minutes and then overnight at 4”, HA and cell fusion activities and the degree of virus flocculation of these samples were determined. 0 - - - 0 HAU/ml, @---a F.T., and 0 . - . C protein content of the virus flocculates of the samples using nonab sorbed anti-XDV serum. a - ~- - L HAL/J~, A---A F.I., and A . - . A protein content of the virus flocculates of the samples using antiNDV serum absorbed with CAF components.
harvest.ed. This procedure of absorption with CAF components was repeated twice. As a control, antiserum alone was ccntrifuged at 105,500 g. One-milliliter aliquots of these antiseruln samples, serially diluted twofold with Hanks’ solution, were mixed in test, tubes with an equal volume of HV,J of a constant concentration of 10,240 HAU/ml. The mixtures were incubated at 37” for 1 hour and then overnight at 4”. Then, the HA and cell fusion activities of these samples were dctermined. As shown in Fig. 4, in the expcrimental series in which control anti-NDV serum was used, the HA titer decreased as the degree of virus flocculation incrcsscd. Enhancement of the cell fusion reaction was observed in some samples. On the contrary, when antiserum absorbed twice with normal CAF components was used, flocculation of
,IXD
TADOKORO
HVJ particles was not apparent, the HA titer did not decrease and the dcgrce of the cell fusion reaction was constant and indcpendent of the amount of serum added. This result clearly indicates t,hat most of the antibody forming a complex with HVJ particles is antibody against a normal component, and that this virus-antibody complex had a high cell fusion activity. MoreOTW. wc have found that, an antiserum agsinst normal chorioallantoic membrane was also capable of enhancing the cell fusion reartion and dccrcasing the HA titer of the virus.
In the sample containing a largcx dose of anti-NDV serum, the cell fusion activity, and the HA activity becrur~ very low, as shown in Figs. 3 and 4. Tl1is might, be due to the fact, that virus flocculatcts grew into very large clumps and w-cnre far fewer in number &an the ETC used for the cell fusion reaction or than red blood cells used for HA titration. Hemozd
of the .4ntiDody against the Host
To st,udy the role of the antibody against the host component in anti-HV,J serum, a 2-1111aliquot of anti-IIV,J serum was ab~orbcd twice with normal CAF cornponcnts l1rcparcd from 50 embryonated eggs. It w::s found that the cell fusion enhancing activity, the HA neutralizing activity: and tlrc HVJ flocculating activity of the antiserum rcmaincd essentially the same as that of nontreated control antiserum, as indicated in Fig. 5. Immunological tests were then made to dctcrmine whether the antibody against the host component in the antiserum was rcmoved by the absorption procedure. A gel diffusion method was employed, using antigens prepared by Hosaka’s method (Hosaka et al., 190). Concentrated HVJ was disintegrated with ether in the presence of Emasol (a nonionizing detergent like Tween 80) and centrifuged at 2000 rpm for 5 minutes; the water phase was separated from the ether phase and used as the HV,J antigen. As normai host antigen, the sedimentable components tlicnterl
in normal CAF were used. As inin Plate 2, six precipitation lines
ASTISERUM
EFFECT
ON CELL
appeared between the HVJ antigen and the control antiserum. These lines were arbitrarily numbered as 1, l’, 2, 5, J$, and 5, respectively, in order from the one nearest to the HVJ antigen. Lines nos. 2 and 5 did not appear between the HVJ antigen and the reabsorbed antiserum. Two lines appeared between the normal CAF component and the control antiserum, but not between the normal component and the reabsorbed antiserum. One line appeared between the control serum and the reabsorbed serum, and t’his seemed to be the same as line no. 2 and a line between the normal component and the control serum. This indicates that the antigen corresponding to line no. 2 was common to HVJ and the normal host component, that the antibody had been completely removed from the reabsorbed antiserum, and that a remnant of the added antigen remained in the serum. The other line between the normal component and the control antiserum may correspond to line no. 5, but this did not appear between HVJ ant.igen and the reabsorbed antiserum or the normal component and the reabsorbed antiserum. It is therefore concluded that antibodies against the normal components in the ant,i-HVJ serum were removed by reabsorption w&h normal CAF components. When t’he anti-HVJ serum was absorbed with KDV particles prepared from infected CAF, the cell fusion enhancing, the HA and the HVJ flocculating neutralizing, actirit,ies of the antiserum did not change. Only when the antiserum was absorbed with HVJ particles, did all these activities disappear, as indicated in Fig. 5. From these findings it is concluded that the antibodies against the normal host components in the anti-HVJ serum had scarcely any significance in the decrease of HA titer, flocculation of the virus, or enhancement of t,he cell fusion activity of the virus. It is the antibodies against the specific HVJ antigens that play a prominent role in these nctivit,ies. Comparison of the Cell Agglutination Activity and the Cell Fusion Activity of HVJ with Antiserum Since the cells are in suspension in the ETC-HVJ system, the first step in cell
FUSION
REACTION
405
HA 5120.. ,. FI. 00 ,’
1260
320,.
6,
4’
80.
2,
20-
o-
FIG. 5. Effects of anti-HVJ serum absorbed with normal CAF components, or with HVJ particles, on HVJ. Anti-HVJ serum, absorbed with sedimentable components in normal CAF, or with purified HVJ particles, or a nonabsorbed control, were mixed with an equal volume of HVJ (10,240 HAU/ml) After incubation at 3” for 60 minutes and then overnight at -1”, the HA and cell fusion activities and the degree of virm flocculation of these samples Here determined. 0 - - - 0 HAU/ ml, @---a F.I., and (> - (> protein content of the virus flocculates of the samples using nonabsorbed anti-HVJ serum. n - - - n HAU/ml, A--A F.I., and A . - A protein content of the virus flocculates of the samples using antiserum absorbed with CAF components. V - - - V HAL/ml, v---‘I F.I., and V . - V protein content of the virus flocculates of the samples using antiserum absorbed with HVJ particles.
fusion is contact between participant cells. This step is caused by the HA activity of HVJ. When ETC and HVJ were mixed, the virus particles were promptly adsorbed onto the surface of the cells and then these cells agglutinated together. The degree of cell fusion after completion of the reaction at 37” may be influenced by the extent of clumping of the cells produced by HVJ. This clumping increases with the number of the virus particles added to ETC. The succeeding steps in the fusion reaction occurred when the cell clump was incubated at 37”, some cells in the clump fused together and the others became dispersed, liberating free cells into the medium. The factor affecting the reaction in these latter
PLATE 2 analysis of anti-HVJ serum absorbed with normal CAF components by the gel diffusion mc(hwl. .41, HVJ an&n (disintegrated with ether); A2, normal C-IF components; El, anti-HVJ serum (unabsorbed); B2, anti-HVJ serum (absorbed with sedimentable components in normal CAF).
al-7
ANTISERUM
EFFECT
ON CELL FUSION REACTION
steps is the cell fusion activity of t,he virus adsorbed onto the cells, unlike the HA activity (Okada and Tadokoro, 1962). A study was made of whether the enhancement of the cell fusion reaction on addition of antiserum is due to an increase in the degree of cell agglutination or to a reinforcement of the cell fusion activity of HVJ. Aliquots of 1 ml of anti-HVJ serum, diluted serially twofold with Hanks’ solution, and an equal volume of HVJ containing 10,240 HAU/ml were mixed and kept ::t 37” for 60 minutes and then overnight at, 4”. Then, 0.5 ml of the mixture of virus and antiserum and an equal volume of ETC suspension containing 1.2 X 10’ cells/ml A.I.
F. I.
407
were mixed in two series of tests tubes at 4”. After standing for 15 minutes at 4”, the tubes of the one series were gently agitated and the samples were introduced into a Fuchs-Rosenthal cytometer and photographed at low magnification. The total number of free cells and cell clumps in the samples were counted from the photographs. The cell agglutination index (A.I.) was calculated as; A.I. =
cell number of nonvirus control tube -1 total number of free cells plus cell clumps in test tube
The other series of tubes was incubated for 60 minutes with shaking at 37” and then their fusion indexes mere determined. The results are shown in Fig. 6. The cell agglutination activity was highest in the control virus sample without antiserum and decreased as the concentration of the antiserum was increased. On the contrary, the cell fusion activity increased in some samples containing a low dose of antiserum. This indicates that the enhancement of the cell fusion reaction is due to an increase of the cell fusion activity of HVJ by the addition of the antiserum, and is not due to an increase of the degree of cell agglutination. DISCUSSION
Antiserum
dilution
factor
FIG. 6. Comparison of cell agglutination activity and cell fusion activity of mixtures of HVJ and anti-HVJ serum. An HVJ suspension (10,240 HAU/ ml) and a serially diluted anti-HVJ serum were mixed in equal volume and incubated for 60 minutes at 37” and then overnight at 4”. One-half milliliter of the mixtures and an equal volume of ETC suspension were then mixed in two series of test tubes. The degree of cell agglutination (A.I.) at 4” was determined from the one series of tubes, and the degree of cell fusion (F.I.) after incubation at 37” for 60 minutes was estimated from the other series of tubes. A - -- A cell agglutination index at 4”; O-O cell fusion index after incubation at 37”.
From the results of previous investigations (Okada, 1962a; Okada and Tadokoro, 1962), it may be concluded that an active substance for the cell fusion reaction is present on the surface of the HVJ particle, and the cell fusion reaction is initiated by a direct reaction of this active substance on the virus with the surface of the participant cells, not by virus infection. The cell fusion reaction in the ETC-HVJ system may progress as follows. The first step is the adsorption of virus particles onto the cells, followed by the agglutination of the cells. The second step is a kind of loosening or disconnecting of the surface structure of the participant cells at 37”. The third step is a connection of the loosened parts of neighboring cell surfaces. If there is no neighboring cell, the loosened site on the cell surface may reconnect with the
408
OKADA,
YAMADA,
main part of the cell. The fourth step is a mixing of the cytoplasm of the participant cells and a formation of a spherical polynuclear cell. The first step in this series of reactions is caused by the HA activity of the virus at 4”. The cell fusion activity of the virus may be involved in the second and third steps. The effect of a large titer of anti-HVJ serum during the cell fusion reaction suggests that at 37” the second and third steps arc complete within 5 minutes from the beginning of the fusion reaction. The fourth step seems to be independent of the activities of the virus. A characteristic of the cell fusion reaction is the necessity for adsorption of many virus particles onto one cell. Thus 1300 virus particles must be adsorbed onto one cell for a 50% decrease in cell number due to fusion of cells (F.I. = 1.0) under good experimental conditions and the degree of the cell fusion reaction increases with the number of adsorbed virus particles (Okada and Tadokoro, 1962). The correlation of the number of adsorbed virus particles with t.he degree of cell fusion may be replaced by a relationship between the density of virus particles per unit of cell surface and the degree of cell fusion. Moreover, only virus particles present between parts of two or more cells which are in contact may participate in the actual cell fusion reaction, and the density of the virus (or the active cell fusion substance of the virus) at this position may be correlated with the efficiency of the change from free to fused cells (or the completion of the second and third steps of the cell fusion reaction). When anti-NDV serum’ was used, the antigen-antibody complex only formed between antigen molecules of host components associated with HVJ particles and antibodies against their antigens, and the complex had a high fusion activity. In this case, the cell fusion activity of the virus in the flocculate may not be neutralized. When virus particles aggregated by anti-NDV serum are adsorbed onto the cells, the adsorption of these aggregates by the free cells may occur. In this case, the density of the active cell fusion substance of the virus aggregate at the part in contact with the cell is higher than when the same
AND TADOKORO
number of free virus particles are adsorbed at random onto the cell surfaces when no anti-NDV serum is present. This phenomenon may be one of the chief reasons why the cell fusion reaction is enhanced by the addition of anti-NDV serum. The increase in cell fusion on addition of anti-HVJ serum, may be due to the same phenomenon, although the activity was not removed by absorption with normal CAF components. If the relative amount of antibodies against virus specific antigens is very much greater in anti-HVJ serum than the amount of antibodies against normal host components, the total activity corresponding to flocculation of HVJ in the serum may be affected very little by removal of antibodies against host components. Moreover, if antibody against the substance causing cell fusion activity is present in small amounts in anti-HVJ serum, the virus flocculation may be produced by combination of antigens and antibodies other than those corresponding to the cell fusion activity. Thus, in this case, the cell fusion activity of the virus in the flocculate might not be neutralized, as in the case with heterologous antiserum, under conditions when the cell fusion reaction was enhanced. If so, the substance showing cell fusion activity in the virus particle may consist of components with a low antibody response. In the HVJ and anti-HVJ serum system, neutralization of the cell lytic activity (HL activity) of the virus with the antiserum may also play a role in the phenomenon of enhancement. Other factors involved in the enhancement phenomenon, such as the influence of the antiserum itself, must also be considered. Normal rabbit serum did not have any affect on the reaction. Ishizaka et al. (1962) indicated that antibody molecuIes, brought into apposition by antigen, interact with each other. The antibody antigen interaction and/or consequent changes in the antibody molecules might be responsible for various biological phenomena. In our experiments, the antibodies which formed a complex with HVJ particles would also acquire
a new biological
activity,
as shown
by Ishizaka et al. To determine whether antibody molecules which are altered by
ANTISERUM
EFFECT
ON CELL
combination with HVJ particles play a role in the cell fusion reaction, the cell fusion reaction was measured using the NDV and anti-NDV complex, because our NDV strain had cell agglutination but not cell fusion activity (Okada and Tadokoro, 1962). The NDV and anti-NDV serum complex, like NDV alone, had no cell fusion activity. In another experiment, a distribution of cell strains or cells showing cell fusion by HVJ was determined and a tendency was observed for undifferentiated cells, but not differentiated celhs, to have a high cell fusion capacity (Okada and Tadokoro, 1963). In the experimental series, leukemia cells from a pat,ient with myeloic leukemia were fused by HVJ, whereas mature polymorpholeucocytes from a patient with rheumatic fever were not. The efficiency of the cell fusion reaction with leukemia cells was enhanced with anti-HV.J serum as in the case of ETC, but the polymorpholeucocytes did not fuse on addition of a mixture of HVd and antiserum, as with HVJ alone. ACKNOWLEDGMENTS The authors wish to express their thanks to Dr K. Fukai, Dr. T. Amano, Dr. M. Yoneda, and Dr. Y. Hosaka of this Institute for their advice. Thanks are also expressed to Dr. M. Shimoyama and Miss F. Murayama for technical assistance. REFERENCES COWIE, D. B., SPIEGELMAN, S., ROBERTS, R., and DUERKSEN, J. D. (1961). Ribosome-bound pgalactosidase. Proc. Natl. Acad. Sci. U.S. 47, 114122. GRANOFF, A., and HENLE, W. (1954). Studies on the hemolytic activity of Newcastle disease virus.
J. Immunol. 72,322-328. HOSAKA, Y., HOSOKAWA, Y., and FUKAI, K. (1960).
FUSION
REACTION
409
Structure of HVJ. I. Two kinds of subunits of HVJ. Bike,l’s J. 3, 27-40. ISHIZAKA, Ii., 1~~1z.4ri.4, T., and SUGAHARA, T. (1962). Biological activity of soluble antigenantibody complexes. VII. Role of an antibody fragment in the induction of biological :tctivities. .I. Itnm unol. 88, 690-701. KSIGTIT, C. A. (1945). Precipitin reactions of highly purifit~d influenza virusIs and related matrrinls. J. Exptl. Med. 82,281-294. Low-su, 0. H., ROSEBROXH, N. J., FARR. L., and R.~XDALL, R. J. (1951). Protein measurement with the Folin phenol reagent, .I. BioZ. C’hc,~l. 193, 265-275. OK;ADA, Y. (1958). The fusion of Ehrlich’s tumor cells caused by HV-J virus itl vifTo. Bikcn’s J. 1, 103-110. OKADA, Y. (1962n). Analysis of giant polgnuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. I. Microscopic observation of giant polynuclrar cell formation. Ex7jtl. Cell Res. 26,98-107. OKAD.~, Y. (196213). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. III. Relationship between cell condition nntl fusion reaction or cell degeneration reaction. Ezptl. Cell Res. 26, 119128. OK.~D.~, T., and TADOICORO,J. (1962). .~nnlysis of giant polynuclrar cell formation caused by HVJ virus from Ehrlich‘s ascitcs tumor cells. II. Quantitative analysis of giant polynuclr::rr cell formation. Exptl. Cell R(a.s. 26, 108-118. OKADA, Y., and T.~DOIWRO, J. (1963). The distribution of cell fusion capacity among several cell strains or cells caused by HVJ. Ez(jf/. (‘c2l 1L’e.s. 32(3!, the December issue. OKADA, Y., SWXJKI, T., and HOSAKA, T. (1957). Interaction between influenza virus and Ehrlich’s tumor cells. III. Fusion phenomenon of Ehrlich’s tumor cells by the action of HVJ, Z strain. Med. J. Osaka Univ. 7,709-717. OKADA, T., NISHIDA, S., and TADOBORO, J. (1961). Correlation between the hemagglutination titer and the virus particle number of HVJ. B&en’s J. 4, 209-214.