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Studies on the soluble antigen of influenza virus
VIROLOGY2, 772-781 (1956)
Studies
on the Soluble
Antigen
of Influenza
II. A Comparison of the EfFects of Sonic Vibration Ether Treatment FLORENCE Division
of Elementary
S. LIEF
AND WERNER
Virus and
Bodies’ HENLE
of Virology,
The Department of Public Health and Preventive Medicine, School of Medicine. The University of Pennsylvania, and The Children’s Hospital of Philadelphia, Pennsylvania Accepted September 6, 1966
A comparison was made of the effects of sonic vibration and of exposure to ether on the release of S antigen from influenza virus. Elementary body suspensions (EB) obtained by high-speed centrifugation or by one cycle of adsorption onto and elution from red cells frequently reacted directly with anti-S sera. The presence of such “external” S depended upon the length of in ovo incubation and upon the strain of virus employed. Following sonic vibration the S titers increased in the EB preparations which initially revealed some S activity, and antigen became detectable in those originally free of measurable quantities. After sonic vibration or even only after second adsorption-elution cycles, the EB suspensions, as a rule, failed to react with anti-S. Although the data indicate that sonic vibration liberated some internal S, its release was not immediately accompanied by detectable losses in infectivity nor was a change noted in the degree of sedimentation of the virus particles by high-speed centrifugation. In any event, exposure to ether proved to be a more potent means of releasing internal S than sonic vibration since no additional S was liberated from ether-treated virus by the latter technique, whereas, in contrast, sonically vibrated EB suspensions on treatment with ether yielded apparently as much S as untreated virus particles. The implications of these results have been discussed. INTRODUCTION
et al., 1946; Kirber and Henle, 1950), In previous reports (Wiener it had been stated that intense sonic vibration of virus suspensions released some S antigen from the elementary bodies without affecting their infectious property. This led to the suggestion that S was on the surface possibly as a contaminant and not an integral part of the virus. 1 The work described the National Institutes
in this paper has been supported by a grant-in-aid of Health, United States Public Health Service. 772
from
EXTERNAL
AND
INTERNAL
S .‘.NTIGES
773
On the other hand, Hoyle (1950, 1952) reported that S antigen could released from within virus particles by their exposure to ether. Since the elementary body suspensions (EB) used by Hoyle reacted per SC with anti-S, his interpretation remained open to question. The data presented in the preceding paper (Lief and Henle, 1956a) on the release of S on exposure to ether of EB suspensions, which were free of directly detectable S, demonstrated more clearly that soluble antigen forms part of the virus particle. This conclusion is further supported by the facts that (a) nearly constant amounts of S can be released per H&4 unit of infectious virus and (b) there are marked changes in the centrifugal sedimentation of treated elementary bodies which indicate either breakdown of the virus or release of heavy components so that the unit’s become less dense. It is the purpose of this communication to present further data on the separation of “external” and ‘Ynt,ernaI” S.
All the general techniques have been described in paper I of this series (Lief and Henle, 1956a). Ether treatment. A standard technique has evolved from the experiments recorded in the preceding paper. Elementary body (EB) suspensions of known hemagglut’inating activity, usually exceeding 5120 HA units per milliliter, were mixed at room t’emperature with 35 volume of Squibb’s anesthetic ether and continuously agitated on a magnetic stirrer so that the ether was well interspersed in the virus suspension. After l-2 hours of treatment, the mixtures were transferred to separatory funnels and the aqueous phases slowly withdrawn. Residual ether was removed by bubbling nitrogen through the material. The HA4 and S fractions were then separated by adsorption of t’he former onto red cells and subsequent elution t’herefrom. Sonic vibration. EB suspensions prepared by either the adsorptionelution t’echnique (one cycle) or by centrifugation were subjected to intense sonic vibration in the treatment vessel of a magneto-striction oscillator (manufactured by the Raytheon Manufacturing Company, Boston, Mass.) for various periods of time. For each period of exposure a constant volume of a given virus preparation was employed t,o eliminate differences in effectiveness due to variation in the amount of liquid. The treated suspensions were separated into HA and S fractions in the same manner as described for fractionation or etaher-treat,ed element,ary bodies.
774
F.
S. LIEF
AND
‘W.
HENLE
EXPERIMENTAL
The e$ect of sonic vibration on elementary body suspensions. A number of EB preparations were obtained from allantoic fluids infected with the PR8 strain by high-speed centrifugation and these were subsequently exposed to sonic vibration for periods of up to 5 hours. The results of representative tests are shown in Table 1. It is seen that the ratio between the hemagglutinin titers with chicken red cells (CHA) and the V antigen (CHA/V) were rather low; i.e., about 8 in contrast to the observations made with EB suspension prepared by the adsorptionelution technique which showed ratios of the order of 32 (Lief and Henle, 1956a, 1956b). It is possible that the addition of RDE for rapid elution of virus may cause the inactivation of inhibitors of hemagglutination, which would remain available in centrifugates. It is also conceivable that insufficient redispersion of virus particles after centrifugal sedimentation lowers the CHA titers without affecting the V antigen however, should have resulted in complete assay. Sonic vibration, dispersion, yet no changes in CHA levels were noted in most experiments. If anything, the titers rather declined slightly on prolonged exposure, as for instance, in experiment number 3 presented in the table. The V antigen activity, likewise, was rarely affected and showed, TABLE EFFECT
OF
SONIC
S0l-k
Exyyt
Preparation
1
ON VARIOUS PROPERTIES INFLUENZA VIRUS
VIBRATION
vibration hours
Hemagglutinin units/ml
IDso/ml lw
CHA
OF
Complem~~~,f~;g -
antigen
V
S
1
EB” SEB
0 2
-
2560 2560
320 320
2
EB SEB
0 2
-
5120 5120
640 640
20 80
3
EB SEB SEB SEB SEB SEB SEB
0 35 1 2 3 4 5
10.3 10.4 10.5 10.2 8.2 7.5 7.4
5120 5120 5120 2560 2560 2560 2560
640 640 640 320 320 320 320
80 80 80 160 160 160 160
a The EB suspensions
were prepared
by high-speed
centrifugation.
EXTERNAL
AND
INTERNAL
S ANTIGEN
775
at most,, slight decreases on prolonged vibration. The original EB SUSpensions did not regularly reveal the presence of detectable S antigen in that the levels varied from < 10 to 80 units per milliliter. In every instance, sonic vibration increased the titers erent,ually from two- to more than fourfold. Such increases were seen before a significant decline in infectivity became apparent (experiment 3). On separation of the HA and S fractions, not shown in the table, all \’ act’ivit,y was found in the former and most of the soluble antigen in the latter. Small quantities of S remained, however, with the hemagglutinins, as was noted also in separat,ion of the corresponding fractions following treatment3 of EB suspensions with ether (Lief and Henle, 1956a). No significant differences were noted in the sedimentation of sonically vibrated virus particles in t#hat the supernates obtained following centrifugation at 25,000 rpm for 30 minutes contained about 10% of the CHA and V activities, regardless of the length of treatment. The remainder of these properties was largely recovered in the sediments. Comparison of sonic vibration and exposure to ether. The above experiments revealed that sonic vibration is capable of removing appreciable quantities of S from elementary body suspensions. It was of interest to compare this t,echnique with the effects of exposure of virus part’icles to et,her. Of several experiment,s two mere chosen for presentation in Table 2. In the first, an EB suspension was prepared by high-speed centrifugation and divided into t,hree parts. One was not treated (EB) ; the second was exposed to ether (EEB); and the third was sot:ically vibrated for 2 hours (SEB). All t,hree preparat,ions were separat’ed into the HA and S fractions by the usual technique. Aliquots of the HA fract’ions of the EB and SEB series were then exposed to et’her and t#he aqueous phases were used for analysis. Part of the HA fraction obtained in the EEB series was in addition sonically vibrated for 2 hours. 1-1scan be seen, the untreated EB preparation was free of directly detectable S and exposure to ether of the HA fraction derived therefrom yielded 160 S antigen units per 5120 CHA units. Thus the CHA/S ratio was 32. The EEB material obtained on treatment of the original EB preparation also contained 160 S units so that the CHA,/S rat,io, likewise, was 32. The HA fraction separated from EEB did not react with anti-S and subsequent sonic vibration failed to release addit’ional quantities of S. Examination of the SEB preparation revealed that some S antigen had been liberated by sonic vibration (CHA/S = 128), which could be separated from the HA fraction. However, treatment of these hemagglu-
776
F.
COMPARISON
Jj3: First z -1
-
-
TABLE
2
HENLE
Haagglutinins units/ml CHA GPHA --__-__-
_-
EB SF
None
2
W.
1?reparaSecond tmn Treatment
corn lemerit P.xmg antigens units/ml V
S
5120 10240 640
--
-
AND
OF SONIC VIBRATION AND EXPOSURE TO ETHER RESPECT TO RELEASE OF S ANTIGEN
Treatment
&i w -
S. LIEF
-
WITH
Ratios
;ym
‘,y
x
CHA/S
8” ---
J$ 36
32
---
x
2c
36 36
4c
4i’
8
>512d
1 x
8
1
64
2
64
1 M
DThe EB suspension was obtained by high-speed centrifugation for the low CHA/V ratio. 6 Hoyle’s technique used (prior to standardization). c Ratio lowered by adverse effect of ether on CHA. d Based upon CHA of original EB.
32d 3Y
128 170
--
---~ 32
32
256 341 64
which may
account
tinins with ether released 160 S units; i.e., an amount indistinguishable from that obtained from previously untreated EB preparations and the CHA/S ratio again was 32. This experiment was carried out prior to the standardization of the ether technique (Lief and Henle, 1956a) and,
EXTERNAL
AND
INTERNAL
777
S ANTIGES
therefore, marked effects of the solvent were noted with respect to losses in HA and V activities. Yet, it is evident that the ratio between HA levels as determined by chicken or guinea pig red cells (CHA/ GPHA) was not affected by sonic vibration and only exposure to ether reduced this ratio. The same held true in the second experiment. Here, the original EB suspension showed some S on direct examination and TABLE THE PRESENCE REMOVAL Experimerit number
1
OF S ANTIGEN BY SECOND CYCLES
Strain of virus
?i%es9f
PRS
20 hrs.
PR8
48 hrs.
PR8
3
72 hrs.
Melbourne
20 hrs.
AND ITS ELUTION
CHA
GPHA
Complement fixing antigen units/ml __-~-~ V s
10240 <5 10240 .__~
-. 15360 <5 10240 -____.
20 240 <20 240 ______
All. Fluid EB, SF HAF (EBT)
10240 <5 10240
15360 <5 7680
30 320 <20 240
30 80 80 <20
All. Fluid EB, SF HAF (EBz) All. Fluid
5120 <5 5120 80
10240 <5 10240 40
(EBg)
10240 10 5120
5120 10 2560
20 60 240 30 <20 20 160 --<20
Hemagglutinins units/ml
Preparation
All. Fluida EBI SF HAF (EB2)
EB, SF HAF
p After
3
IN EBI PREPARATIONS OF ADSORPTION AND
-
La47
20 hrs.
All. Fluid E& SF HAF (EBy,)
160 2560 <5 5120
160 5120 <5 5120
Philadelphia 1951
20 hrs.
All. Fluid EBI SF HAF (EB2)
160 20480 <5 10240
160 10240 10 10240
10 240 240 <20
absorption
with
chicken
red cells.
778
F.
S. LIEF
AND
W.
HENLE
its titer increased as a result of sonic vibration. The separated HA fraction no longer reacted with anti-S but on treatment with ether under standard conditions, again substantial quantities of S were liberated. The CHA/A ratio was 64; i.e., within the limits of that of standard virus preparations (Lief and Henle, 1956a, 1956b). Removal of S from EB1 preparations. As was recorded above and in the preceding paper of this series, elementary body suspensions prepared by either high-speed centrifugation or by one cycle of adsorption onto and elution from red cells (EBI) often revealed directly detectable quantities of S antigen. The presence of S in such preparations appeared to be related to (a) the period of in ovo incubation employed for the production of virus; and (b) the strain of virus used. This was borne out in further experiments. If EB1 preparations of the PR8 strain were derived from allantoic fluids collected 20 hours following inoculation they rarely contained detectable S; if obtained from allantoic fluids after longer incubation they regularly revealed some S activity. This is shown, for example, in experiment number 1 of Table 3. Previous studies (Kirber and Henle, 1950) employing similar quantities of seed virus as inocula, showed the absence of free S antigen in allantoic fluids after incubation for 24 hours. In later harvests substantial titers were found. This was confirmed in the experiments presented in Table 3. In the second experiment recorded in the table, results are summarized with EB1 preparations of the Melbourne, L347, and Philadelphia 51 strains, all derived from 20-hour harvests. While in this instance the Melbourne EBI suspension contained S, in other experiments it was found free of detectable S. With respect to the other two viruses, both A prime strains, it has not been possible to obtain EB1 suspensions free of soluble antigen under the conditions described. It is evident, however, that whatever S was present in any one of the EB1 preparations was apparently not firmly bound to the elementary bodies. As shown in Table 3, on second adsorption of the virus onto chicken red cells the S antigen remained behind (SF). The eluted hemagglutinins (HAF), which correspond to EB2 suspensions, now failed to react with anti-S; yet on exposure to ether these yield substantial quantities of S (Lief and Henle, 1956a). DISCUSSION
Past experiments indicated that elementary bodies derived by centrifugation from allantoic fluid of chick embryos infected 48 hours pre-
EXTERNAL
AND
INTERNBL
S ANTIGEN
779
viously with various influenza strains contained some S antigen on the surface (Wiener et al., 1946; Kirber and Henle, 1950). This suggestion was supported by the facts that such EB suspensions, when used in sufficient concentration, reacted with anti-S in complement fixat’ion tests or absorbed anti-S as well as anti-V from convalescent sera. If such preparations were sonically vibrated and the virus part,icles, which remained infectious, were sedimented thereafter by high-speed centrifugation, the supernates contained S antigen and the resuspended sediments failed to react with or to absorb anti-S. The present data confirm the existence of such externally located S. It was found that the presence or absence of S antigen in preparations obtained either by high-speed centrifugation or by a single cycle of adsorption onto and elution from red cells depended upon the period of incubation of the virus in ovo and upon the strain of virus employed. Such EB suspensions of PR8 or Melbourne viruses, prepared from 20-hour harvests, were generally free of directly detectable S, whereas those obtained after longer incubation in ovo regularly revealed S activity. With more recently isolated strains (L347 and Philadelphia 1951) even 20-hour EB1 preparations always contained S. Following sonic vibration of such suspensions S could readily be separated by the adsorption-elution technique from the HA components and these often no longer reacted with anti-S. However, merely subjecbion of an unt,reated aliquot of such EB suspensions to a cycle of adsorption onto and elution from red cells also led frequently to separation of these S components. Thus, sonic vibration would not always seem to be essential for removal of external S. Yet, it appears to be evident that the antigen so removed was attached originally to the elementary bodies because it, followed bhe virus through the first adsorption-elution or sediment,at.ion-resuspension cycles, It is unlikely that the antigen found represented merely free S, which was trapped in the RBC-virus complexes or in the sediments, since the total amount in the EB suspensions oft,en exceeded by far that found in the allantoic fluids after removal of the virus particles. The data presented indicate in addition that sonic vibration may release some int,ernal S. If EB suspensions, which contained detectable S, were sonically vibrated for more than one hour t.he titers increased, or, if preparations were treated, which appeared to be free of S, appreciable quantities of S became detectable. These effects were noticeable even before significant losses in infectivity were noted. It must be realized,
780
F.
S. LIEF
AND
W.
HENLE
however, that the technique employed for IDbo titrations is accurate only within 0.6 log10 units (Knight, 1944) and thus inactivation or breakdown of a considerable proportion of the virus particles may have escaped detection. On the other hand, sonic vibration did not affect the sedimentation of the elementary bodies by high-speed centrifugation in a significant manner in contrast to the observations made with ethertreated virus (Lief and Henle, 1956a). It is apparent from these results that sonic vibration is inefficient as a means of releasing internal S when compared to the results obtained on exposure to ether. This conclusion is further supported by the fact that sonic vibration of ether-treated virus failed to yield any additional S. In contrast, elementary bodies which had been sonically vibrated for as long as 5 hours yielded apparently as much S antigen on treatment with ether as did standard virus preparations, in that the CHA/S ratios fell between 32 and 64. These data resolve differences in interpretation of experimental results. Fulton (1949) and Kirber and Henle (1950) suggested that S represented a surface contaminant and was not an essential component incorporated into the virus. The results of sonic vibration presented here and previously (Wiener et al., 1946; Kirber and Henle, 1950) as well as those obtained with 2 cycles of adsorption and elution show that this in part is true. Hoyle (1952), on the other hand, proposed that S forms an essential part of the virus based on the effects of exposure of virus to ether, resulting in the release of considerable quantities of S. However, the EB suspensions of the DSP strain employed by Hoyle always showed some S activity prior to treatment which to some extent confused interpretation of the results. The fact, established here and in the preceding paper, that EB suspensions free of detectable S activity also yielded large quantities of S on exposure to ether provides a firmer basis for the view that S forms an integral part of the virus particle. Such internal S remains undetectable until liberated by the solvent. This interpretation is further strengthened by the observations that standard virus contains fairly uniform quantities of S (Lief and Henle, 1956a) and that in EB preparations obtained under conditions favoring incomplete virus formation, the amount of S released decreases with increasing incompleteness of the population (Lief and Henle, 1956b). REFERENCES FULTON, F. (1949). Growth cycle of influenza virus. Nature 164, 189-190. HOYLE, L. (1950). The multiplication of influenza viruses in the fertile egg. J.
Hyg. 48, 277-297.
EXTERNAL
HOYLE, L. (1952). Structure
AND
INTERNAL
S ANTIGEN
781
of influenza virus. The relation between biological and chemical structure of virus fractions. J. Hyg. 60, 229-245. KIRBER, M. W., and HENLE, W. (1956). A comparison of influenza complement fixation antigens derived from allantoic fluids and membranes. J. Immunol. 66, 229-244. KNIGHT, C. A. (1944). Titration of influenza virus in chick embryos. 6. EzptE. Med. 79, 487495. LIEF, F. S., and HENLE, W. (1956a). Studies on the soluble antigen of influenza virus. I. The release of S antigen from elementary hodies by treatment with ether. Virology 2,753-771. LIEF, F. S., and HENLE, W. (1956b). Studies on the soluble antigen of influenza virus. III. The decreased incorporation of S antigen into elementary bodies of increasing incompleteness. Virology 2.782-797. WIENER, M., HENLE, W., and HENLE, G. (1946). Studies on the complement fixation antigens of influenza viruses types A and B. J. Exptl. Med. 63, 259-279. activity
Studies
on the Soluble
Antigen
of Influenza
II. A Comparison of the EfFects of Sonic Vibration Ether Treatment FLORENCE Division
of Elementary
S. LIEF
AND WERNER
Virus and
Bodies’ HENLE
of Virology,
The Department of Public Health and Preventive Medicine, School of Medicine. The University of Pennsylvania, and The Children’s Hospital of Philadelphia, Pennsylvania Accepted September 6, 1966
A comparison was made of the effects of sonic vibration and of exposure to ether on the release of S antigen from influenza virus. Elementary body suspensions (EB) obtained by high-speed centrifugation or by one cycle of adsorption onto and elution from red cells frequently reacted directly with anti-S sera. The presence of such “external” S depended upon the length of in ovo incubation and upon the strain of virus employed. Following sonic vibration the S titers increased in the EB preparations which initially revealed some S activity, and antigen became detectable in those originally free of measurable quantities. After sonic vibration or even only after second adsorption-elution cycles, the EB suspensions, as a rule, failed to react with anti-S. Although the data indicate that sonic vibration liberated some internal S, its release was not immediately accompanied by detectable losses in infectivity nor was a change noted in the degree of sedimentation of the virus particles by high-speed centrifugation. In any event, exposure to ether proved to be a more potent means of releasing internal S than sonic vibration since no additional S was liberated from ether-treated virus by the latter technique, whereas, in contrast, sonically vibrated EB suspensions on treatment with ether yielded apparently as much S as untreated virus particles. The implications of these results have been discussed. INTRODUCTION
et al., 1946; Kirber and Henle, 1950), In previous reports (Wiener it had been stated that intense sonic vibration of virus suspensions released some S antigen from the elementary bodies without affecting their infectious property. This led to the suggestion that S was on the surface possibly as a contaminant and not an integral part of the virus. 1 The work described the National Institutes
in this paper has been supported by a grant-in-aid of Health, United States Public Health Service. 772
from
EXTERNAL
AND
INTERNAL
S .‘.NTIGES
773
On the other hand, Hoyle (1950, 1952) reported that S antigen could released from within virus particles by their exposure to ether. Since the elementary body suspensions (EB) used by Hoyle reacted per SC with anti-S, his interpretation remained open to question. The data presented in the preceding paper (Lief and Henle, 1956a) on the release of S on exposure to ether of EB suspensions, which were free of directly detectable S, demonstrated more clearly that soluble antigen forms part of the virus particle. This conclusion is further supported by the facts that (a) nearly constant amounts of S can be released per H&4 unit of infectious virus and (b) there are marked changes in the centrifugal sedimentation of treated elementary bodies which indicate either breakdown of the virus or release of heavy components so that the unit’s become less dense. It is the purpose of this communication to present further data on the separation of “external” and ‘Ynt,ernaI” S.
All the general techniques have been described in paper I of this series (Lief and Henle, 1956a). Ether treatment. A standard technique has evolved from the experiments recorded in the preceding paper. Elementary body (EB) suspensions of known hemagglut’inating activity, usually exceeding 5120 HA units per milliliter, were mixed at room t’emperature with 35 volume of Squibb’s anesthetic ether and continuously agitated on a magnetic stirrer so that the ether was well interspersed in the virus suspension. After l-2 hours of treatment, the mixtures were transferred to separatory funnels and the aqueous phases slowly withdrawn. Residual ether was removed by bubbling nitrogen through the material. The HA4 and S fractions were then separated by adsorption of t’he former onto red cells and subsequent elution t’herefrom. Sonic vibration. EB suspensions prepared by either the adsorptionelution t’echnique (one cycle) or by centrifugation were subjected to intense sonic vibration in the treatment vessel of a magneto-striction oscillator (manufactured by the Raytheon Manufacturing Company, Boston, Mass.) for various periods of time. For each period of exposure a constant volume of a given virus preparation was employed t,o eliminate differences in effectiveness due to variation in the amount of liquid. The treated suspensions were separated into HA and S fractions in the same manner as described for fractionation or etaher-treat,ed element,ary bodies.
774
F.
S. LIEF
AND
‘W.
HENLE
EXPERIMENTAL
The e$ect of sonic vibration on elementary body suspensions. A number of EB preparations were obtained from allantoic fluids infected with the PR8 strain by high-speed centrifugation and these were subsequently exposed to sonic vibration for periods of up to 5 hours. The results of representative tests are shown in Table 1. It is seen that the ratio between the hemagglutinin titers with chicken red cells (CHA) and the V antigen (CHA/V) were rather low; i.e., about 8 in contrast to the observations made with EB suspension prepared by the adsorptionelution technique which showed ratios of the order of 32 (Lief and Henle, 1956a, 1956b). It is possible that the addition of RDE for rapid elution of virus may cause the inactivation of inhibitors of hemagglutination, which would remain available in centrifugates. It is also conceivable that insufficient redispersion of virus particles after centrifugal sedimentation lowers the CHA titers without affecting the V antigen however, should have resulted in complete assay. Sonic vibration, dispersion, yet no changes in CHA levels were noted in most experiments. If anything, the titers rather declined slightly on prolonged exposure, as for instance, in experiment number 3 presented in the table. The V antigen activity, likewise, was rarely affected and showed, TABLE EFFECT
OF
SONIC
S0l-k
Exyyt
Preparation
1
ON VARIOUS PROPERTIES INFLUENZA VIRUS
VIBRATION
vibration hours
Hemagglutinin units/ml
IDso/ml lw
CHA
OF
Complem~~~,f~;g -
antigen
V
S
1
EB” SEB
0 2
-
2560 2560
320 320
2
EB SEB
0 2
-
5120 5120
640 640
20 80
3
EB SEB SEB SEB SEB SEB SEB
0 35 1 2 3 4 5
10.3 10.4 10.5 10.2 8.2 7.5 7.4
5120 5120 5120 2560 2560 2560 2560
640 640 640 320 320 320 320
80 80 80 160 160 160 160
a The EB suspensions
were prepared
by high-speed
centrifugation.
EXTERNAL
AND
INTERNAL
S ANTIGEN
775
at most,, slight decreases on prolonged vibration. The original EB SUSpensions did not regularly reveal the presence of detectable S antigen in that the levels varied from < 10 to 80 units per milliliter. In every instance, sonic vibration increased the titers erent,ually from two- to more than fourfold. Such increases were seen before a significant decline in infectivity became apparent (experiment 3). On separation of the HA and S fractions, not shown in the table, all \’ act’ivit,y was found in the former and most of the soluble antigen in the latter. Small quantities of S remained, however, with the hemagglutinins, as was noted also in separat,ion of the corresponding fractions following treatment3 of EB suspensions with ether (Lief and Henle, 1956a). No significant differences were noted in the sedimentation of sonically vibrated virus particles in t#hat the supernates obtained following centrifugation at 25,000 rpm for 30 minutes contained about 10% of the CHA and V activities, regardless of the length of treatment. The remainder of these properties was largely recovered in the sediments. Comparison of sonic vibration and exposure to ether. The above experiments revealed that sonic vibration is capable of removing appreciable quantities of S from elementary body suspensions. It was of interest to compare this t,echnique with the effects of exposure of virus part’icles to et,her. Of several experiment,s two mere chosen for presentation in Table 2. In the first, an EB suspension was prepared by high-speed centrifugation and divided into t,hree parts. One was not treated (EB) ; the second was exposed to ether (EEB); and the third was sot:ically vibrated for 2 hours (SEB). All t,hree preparat,ions were separat’ed into the HA and S fractions by the usual technique. Aliquots of the HA fract’ions of the EB and SEB series were then exposed to et’her and t#he aqueous phases were used for analysis. Part of the HA fraction obtained in the EEB series was in addition sonically vibrated for 2 hours. 1-1scan be seen, the untreated EB preparation was free of directly detectable S and exposure to ether of the HA fraction derived therefrom yielded 160 S antigen units per 5120 CHA units. Thus the CHA/S ratio was 32. The EEB material obtained on treatment of the original EB preparation also contained 160 S units so that the CHA,/S rat,io, likewise, was 32. The HA fraction separated from EEB did not react with anti-S and subsequent sonic vibration failed to release addit’ional quantities of S. Examination of the SEB preparation revealed that some S antigen had been liberated by sonic vibration (CHA/S = 128), which could be separated from the HA fraction. However, treatment of these hemagglu-
776
F.
COMPARISON
Jj3: First z -1
-
-
TABLE
2
HENLE
Haagglutinins units/ml CHA GPHA --__-__-
_-
EB SF
None
2
W.
1?reparaSecond tmn Treatment
corn lemerit P.xmg antigens units/ml V
S
5120 10240 640
--
-
AND
OF SONIC VIBRATION AND EXPOSURE TO ETHER RESPECT TO RELEASE OF S ANTIGEN
Treatment
&i w -
S. LIEF
-
WITH
Ratios
;ym
‘,y
x
CHA/S
8” ---
J$ 36
32
---
x
2c
36 36
4c
4i’
8
>512d
1 x
8
1
64
2
64
1 M
DThe EB suspension was obtained by high-speed centrifugation for the low CHA/V ratio. 6 Hoyle’s technique used (prior to standardization). c Ratio lowered by adverse effect of ether on CHA. d Based upon CHA of original EB.
32d 3Y
128 170
--
---~ 32
32
256 341 64
which may
account
tinins with ether released 160 S units; i.e., an amount indistinguishable from that obtained from previously untreated EB preparations and the CHA/S ratio again was 32. This experiment was carried out prior to the standardization of the ether technique (Lief and Henle, 1956a) and,
EXTERNAL
AND
INTERNAL
777
S ANTIGES
therefore, marked effects of the solvent were noted with respect to losses in HA and V activities. Yet, it is evident that the ratio between HA levels as determined by chicken or guinea pig red cells (CHA/ GPHA) was not affected by sonic vibration and only exposure to ether reduced this ratio. The same held true in the second experiment. Here, the original EB suspension showed some S on direct examination and TABLE THE PRESENCE REMOVAL Experimerit number
1
OF S ANTIGEN BY SECOND CYCLES
Strain of virus
?i%es9f
PRS
20 hrs.
PR8
48 hrs.
PR8
3
72 hrs.
Melbourne
20 hrs.
AND ITS ELUTION
CHA
GPHA
Complement fixing antigen units/ml __-~-~ V s
10240 <5 10240 .__~
-. 15360 <5 10240 -____.
20 240 <20 240 ______
All. Fluid EB, SF HAF (EBT)
10240 <5 10240
15360 <5 7680
30 320 <20 240
30 80 80 <20
All. Fluid EB, SF HAF (EBz) All. Fluid
5120 <5 5120 80
10240 <5 10240 40
(EBg)
10240 10 5120
5120 10 2560
20 60 240 30 <20 20 160 --<20
Hemagglutinins units/ml
Preparation
All. Fluida EBI SF HAF (EB2)
EB, SF HAF
p After
3
IN EBI PREPARATIONS OF ADSORPTION AND
-
La47
20 hrs.
All. Fluid E& SF HAF (EBy,)
160 2560 <5 5120
160 5120 <5 5120
Philadelphia 1951
20 hrs.
All. Fluid EBI SF HAF (EB2)
160 20480 <5 10240
160 10240 10 10240
10 240 240 <20
absorption
with
chicken
red cells.
778
F.
S. LIEF
AND
W.
HENLE
its titer increased as a result of sonic vibration. The separated HA fraction no longer reacted with anti-S but on treatment with ether under standard conditions, again substantial quantities of S were liberated. The CHA/A ratio was 64; i.e., within the limits of that of standard virus preparations (Lief and Henle, 1956a, 1956b). Removal of S from EB1 preparations. As was recorded above and in the preceding paper of this series, elementary body suspensions prepared by either high-speed centrifugation or by one cycle of adsorption onto and elution from red cells (EBI) often revealed directly detectable quantities of S antigen. The presence of S in such preparations appeared to be related to (a) the period of in ovo incubation employed for the production of virus; and (b) the strain of virus used. This was borne out in further experiments. If EB1 preparations of the PR8 strain were derived from allantoic fluids collected 20 hours following inoculation they rarely contained detectable S; if obtained from allantoic fluids after longer incubation they regularly revealed some S activity. This is shown, for example, in experiment number 1 of Table 3. Previous studies (Kirber and Henle, 1950) employing similar quantities of seed virus as inocula, showed the absence of free S antigen in allantoic fluids after incubation for 24 hours. In later harvests substantial titers were found. This was confirmed in the experiments presented in Table 3. In the second experiment recorded in the table, results are summarized with EB1 preparations of the Melbourne, L347, and Philadelphia 51 strains, all derived from 20-hour harvests. While in this instance the Melbourne EBI suspension contained S, in other experiments it was found free of detectable S. With respect to the other two viruses, both A prime strains, it has not been possible to obtain EB1 suspensions free of soluble antigen under the conditions described. It is evident, however, that whatever S was present in any one of the EB1 preparations was apparently not firmly bound to the elementary bodies. As shown in Table 3, on second adsorption of the virus onto chicken red cells the S antigen remained behind (SF). The eluted hemagglutinins (HAF), which correspond to EB2 suspensions, now failed to react with anti-S; yet on exposure to ether these yield substantial quantities of S (Lief and Henle, 1956a). DISCUSSION
Past experiments indicated that elementary bodies derived by centrifugation from allantoic fluid of chick embryos infected 48 hours pre-
EXTERNAL
AND
INTERNBL
S ANTIGEN
779
viously with various influenza strains contained some S antigen on the surface (Wiener et al., 1946; Kirber and Henle, 1950). This suggestion was supported by the facts that such EB suspensions, when used in sufficient concentration, reacted with anti-S in complement fixat’ion tests or absorbed anti-S as well as anti-V from convalescent sera. If such preparations were sonically vibrated and the virus part,icles, which remained infectious, were sedimented thereafter by high-speed centrifugation, the supernates contained S antigen and the resuspended sediments failed to react with or to absorb anti-S. The present data confirm the existence of such externally located S. It was found that the presence or absence of S antigen in preparations obtained either by high-speed centrifugation or by a single cycle of adsorption onto and elution from red cells depended upon the period of incubation of the virus in ovo and upon the strain of virus employed. Such EB suspensions of PR8 or Melbourne viruses, prepared from 20-hour harvests, were generally free of directly detectable S, whereas those obtained after longer incubation in ovo regularly revealed S activity. With more recently isolated strains (L347 and Philadelphia 1951) even 20-hour EB1 preparations always contained S. Following sonic vibration of such suspensions S could readily be separated by the adsorption-elution technique from the HA components and these often no longer reacted with anti-S. However, merely subjecbion of an unt,reated aliquot of such EB suspensions to a cycle of adsorption onto and elution from red cells also led frequently to separation of these S components. Thus, sonic vibration would not always seem to be essential for removal of external S. Yet, it appears to be evident that the antigen so removed was attached originally to the elementary bodies because it, followed bhe virus through the first adsorption-elution or sediment,at.ion-resuspension cycles, It is unlikely that the antigen found represented merely free S, which was trapped in the RBC-virus complexes or in the sediments, since the total amount in the EB suspensions oft,en exceeded by far that found in the allantoic fluids after removal of the virus particles. The data presented indicate in addition that sonic vibration may release some int,ernal S. If EB suspensions, which contained detectable S, were sonically vibrated for more than one hour t.he titers increased, or, if preparations were treated, which appeared to be free of S, appreciable quantities of S became detectable. These effects were noticeable even before significant losses in infectivity were noted. It must be realized,
780
F.
S. LIEF
AND
W.
HENLE
however, that the technique employed for IDbo titrations is accurate only within 0.6 log10 units (Knight, 1944) and thus inactivation or breakdown of a considerable proportion of the virus particles may have escaped detection. On the other hand, sonic vibration did not affect the sedimentation of the elementary bodies by high-speed centrifugation in a significant manner in contrast to the observations made with ethertreated virus (Lief and Henle, 1956a). It is apparent from these results that sonic vibration is inefficient as a means of releasing internal S when compared to the results obtained on exposure to ether. This conclusion is further supported by the fact that sonic vibration of ether-treated virus failed to yield any additional S. In contrast, elementary bodies which had been sonically vibrated for as long as 5 hours yielded apparently as much S antigen on treatment with ether as did standard virus preparations, in that the CHA/S ratios fell between 32 and 64. These data resolve differences in interpretation of experimental results. Fulton (1949) and Kirber and Henle (1950) suggested that S represented a surface contaminant and was not an essential component incorporated into the virus. The results of sonic vibration presented here and previously (Wiener et al., 1946; Kirber and Henle, 1950) as well as those obtained with 2 cycles of adsorption and elution show that this in part is true. Hoyle (1952), on the other hand, proposed that S forms an essential part of the virus based on the effects of exposure of virus to ether, resulting in the release of considerable quantities of S. However, the EB suspensions of the DSP strain employed by Hoyle always showed some S activity prior to treatment which to some extent confused interpretation of the results. The fact, established here and in the preceding paper, that EB suspensions free of detectable S activity also yielded large quantities of S on exposure to ether provides a firmer basis for the view that S forms an integral part of the virus particle. Such internal S remains undetectable until liberated by the solvent. This interpretation is further strengthened by the observations that standard virus contains fairly uniform quantities of S (Lief and Henle, 1956a) and that in EB preparations obtained under conditions favoring incomplete virus formation, the amount of S released decreases with increasing incompleteness of the population (Lief and Henle, 1956b). REFERENCES FULTON, F. (1949). Growth cycle of influenza virus. Nature 164, 189-190. HOYLE, L. (1950). The multiplication of influenza viruses in the fertile egg. J.
Hyg. 48, 277-297.
EXTERNAL
HOYLE, L. (1952). Structure
AND
INTERNAL
S ANTIGEN
781
of influenza virus. The relation between biological and chemical structure of virus fractions. J. Hyg. 60, 229-245. KIRBER, M. W., and HENLE, W. (1956). A comparison of influenza complement fixation antigens derived from allantoic fluids and membranes. J. Immunol. 66, 229-244. KNIGHT, C. A. (1944). Titration of influenza virus in chick embryos. 6. EzptE. Med. 79, 487495. LIEF, F. S., and HENLE, W. (1956a). Studies on the soluble antigen of influenza virus. I. The release of S antigen from elementary hodies by treatment with ether. Virology 2,753-771. LIEF, F. S., and HENLE, W. (1956b). Studies on the soluble antigen of influenza virus. III. The decreased incorporation of S antigen into elementary bodies of increasing incompleteness. Virology 2.782-797. WIENER, M., HENLE, W., and HENLE, G. (1946). Studies on the complement fixation antigens of influenza viruses types A and B. J. Exptl. Med. 63, 259-279. activity