Studies on the soluble antigen of influenza virus

VIROLOGY

2,

Studies

753-771 (1956)

on the Soluble

Antigen

I. The Release of S Antigen by Treatment FLORENCE Division School

of Virology,

of Medicine.

S. LIEF

of Influenza

from Elementary with Ether’

AND WERNER

Virus Bodies

HENLE

The Department of Public Health and Preventive The University of Pennsylvania, and The Children’s of Philadelphia, Pennsylvania

Jledicine, Hospital.

Accepted September 5, 1956 Soluble (S) antigen is readily released from influenza virus particles by exposure to ether. In order to obtain reproducible results with maximal yields of S and minimal damage to or loss of hemagglutinating and virus (V) antigen activities the technique described by Hoyle was modified and standardized. Elementary body suspensions which contained at least 5120 HA units per milliliter, and which failed to react per se with anti-S, were exposed to 35 volume of anesthetic ether at room temperature for periods up to 2 hours under constant agitation by a magnetic stirrer. Under these conditions, the hemagglutinating activity, as measured with guinea pig red cells, increased up to sixteenfold with all four influenza A strains tested; agglutination of chicken red cells was slightly enhanced with one of the strains and reduced twofold OI more with the other three. The V antigen levels remained essentially ~11. altered. On the average, about 40 HA units of standard elementary bodies were required to yield one unit of S. Release of 8 became apparent within a few minutes of treatment. This was accompanied by nearly complete loss of infectivity and the sedimentation of the HA activity by high-speed cerltrifugation was markedly reduced. These results do not necessarily imply that the virus pnrticl(h-; arc diailltegrated, as has been discussed. INTRODUCTION

iMany virus infections are accompanied by the elaboration of so-called soluble or S antigens. These are generally smaller in size than t,he virus particles and thus can be readily separated, as a rule, from the elementary bodies by physical techniques. The S antigen of influenza \4rus has 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. 753

from

754

F.

S. LIEF

AND

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HENLE

been studied more extensively than many others. Tissues infected with this agent such as mouse lungs or chorioallantoic membranes of the chick embryo are rich in S antigen. Relatively little however is released along with virus from the infected cells. It is separable from the virus particle by sedimentation of the latter by high-speed centrifugation or by adsorption of the elementary bodies onto red cells. The S antigen which is not readily sedimented and not adsorbed onto RBC possesses only complement fixing activity in the presence of appropriate sera. The virus particles which also contain CF antigens, in addition reveal infective, hemagglutinating, toxic, interfering, and other activities. The S antigen and the bulk of the virus or V antigens are serologically distinct (Henle and Wiener, 1944; Hoyle, 1945; Wiener et al., 1946) because (a) they reveal usually different patterns of activity in optimal titration tests; (b) in carefully controlled serum cross-absorption tests S removes anti-S but not anti-V and vice versa; and (c) post-vaccination sera react usually with V antigen only, whereas convalescent sera will react with both V and S, but the respective antibody titers show no correlation. The virus particles are not always free of detectable S (Henle et al., 1944; Hoyle, 1945; Wiener et al., 1946). This is particularly evident from the experiments of Wiener et al. (1946) and Kirber and Henle (1950), who showed that (a) an anti-S serum (absorbed with elementary bodies) may fail to react with moderate quantities of virus but may do so in the presence of large amounts; (b) absorption of a convalescent serum with virus particles may remove anti-S as well as anti-V; (c) sonic vibration of virus particles shears off S with little effect on infectivity and the centrifugally separated elementary bodies then no longer react with anti-S nor do they remove anti-S when used in large quantities for absorption of convalescent sera; and, finally (d) as reported by Hoyle (1950, 1952) treatment of elementary bodies with ether removes appreciable amounts of S. The soluble antigen is type-specific in that S preparations derived from various influenza A strains were found to be identical but distinct from those of influenza B virus (Lennette and Horsfall, 1941; Henle and Wiener, 1944; Hoyle, 1945; Kirber and Henle, 1950). The V antigens have strong strain-specific components and to some extent also crossreacting antigens within the type (Friedewald, 1943, 1944; Henle and Wiener, 1944; Hoyle, 1945; Wiener et al., 1946). Since, as pointed out above, virus particles may contain some S, strain-specificity is not always apparent when convalescent sera are employed. Cross-reactions

SOLUBLE

ANTIGEN

IN

INFLUENZA

VIM’S

755

between V antigens of different homotypic strains can be considered significant only if observed with sera free of anti-S (Lief, unpublished). The nature of the S antigen as well as its role in the infectious process are still obscure. It has been suggested (Fulton, 1949; Kirber and Henle, 1950) that S represents a product of host-virus interaction, a sort, of matrix substance, but that it does not form an integral part of the virus. In support of this view, several facts were cited; (a) much greater con(‘eiltrations of S are found in the tissues than in elementary body suspensions; (b) S can be removed from the virus by sonic vibrat,iou wit,hout loss of infectivity and hemagglutinating activity; and ((3) vaccines prepared from inactivated virus fail, as a rule, to produce ant,i-S. In contrast, Hoyle (1950, 1952) suggested that, S represents a most’ essential part of the virus, in fact,, the basic self-replicating unit,. His view is based upon the results of treatment of elementary bodies wit,h ether and the events as they occur in the growth cycle of the virus in the allantoic. membrane. Hoyle observed that upon exposure t.o ether of element,ary body preparations which reacted per se with ant’i S (a) t,he infectivity was largely lost; (b) hemagglutination of guinea pig red cells increased; (c) the V antigen activity was markedly reduced or disappeared; (d) S ant)igen was set free and then could readily be separated from t#hr IIA components; and, finally (e) certain chemical analyses indicated the S preparation to be a ribonucleoprotein and t’he separat#ed RX fraction a protein with enzymic properties. Electron micrographs of ether-treated virus revealed particles of varying sizes, all smaller than elementary bodies (Hoyle et al., 1953). On the basis of these results, Hoyle suggested t’hat the virus particles are composed of numerous smaller units having either 8 or HA activity and these are enclosed in a lipid en\-elope. On destruction of the latter by ether these units are released. 111additioll, he advanced the concept that the elementary body on entry ir1t.o a susceptible cell similarly breaks down into the smaller sub-unit,s, liberat,ing soluble antigen which then functions as the basic self-replicaat,ing unit,. This concept seems t#obe supported by suggestive evidence of breakdown of seed virus labeled with P3* (Hoyle and Frisch-Xiggemeyrr, 1955) and t’he appearance of S antigen in the infected tissues prior to the development of V antigen and HA activity (Hoyle, 1948, 1952: Henleand Ht~~le, 1949). While many aspects of Hoyle’s concept are speculative, it, wax felt, that. the efiects of ether treatment alone were of sufficient interest to lnerit furt,her investigation. Many of the observations t,o be reported ill this

756

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series confirm Hoyle’s data but certain discrepancies, capable of &fferent interpretation, were also noted. The first report is concerned with standardization of the ether technique and its application to the study of fully infectious influenza A viruses. MATERIALS

AND

METHODS

Strains of Virus Four strains of influenza virus type A were used: PR8 (1934) ; Melbourne (1935) ; La (1947) ; and Philadelphia (1951). The seeds were prepared by allantoic inoculation of chick embryos with dilute virus and the allantoic fluids collected after appropriate incubation periods were stored in flame-sealed ampules in the dry ice chest. Virus Preparations (a). Preparation of elementary body suspensions from allantoic fluids by adsorption-e&ion technique. Twelve-day old chick embryos were inoculated allantoically with lo6 to lo6 IDso of standard seed virus. After 20 hours of incubation in ovo at 37” bloody allantoic fluids were collected by tearing through the chorioallantoic membrane and allowing free bleeding to occur into the embryonic cavity. The fluids were immediately chilled in an ice bath and kept at 0” for 1 hour. The red cells were sedimented by centrifugation in the cold, washed once with ice-cold M/100 phosphate buffered saline solution at pH 7.0 (BSS) and resuspended in BSS to which had been added 1% RDE (50 units per milliliter), penicillin (500 units per milliliter) and streptomycin (100 pg/ml). For each egg harvested, 1 ml of BSS was employed. Elution of the virus was allowed to take place for 235 hours in a 37” water bath. The red cells were then removed by centrifugation and the supernatant fluid (eluate) constituted the elementary body suspension (EBI). For special experiments second cycles of adsorption and elution were employed (EBz). (b). Preparations qf elementary body suspensions by high-speed centrifugation. Clear allantoic fluids were collected from chilled embryos

which had been inoculated 20 hours previously with lo6 to IO6 EIDSO of standard seed. These were clarified by preliminary centrifugation at 2000 rpm for 10 minutes and then centrifuged at 25,090 rpm for 30 minutes. The pellets before resuspension in BSS were washed with saline solution by carefully allowing the liquid to flow over the surface of the pellet. The quantity of BSS used for resuspension depended on the desired concentration of virus, although usually 1 ml was used per egg

SOLUBLE

ANTIGEN

IN

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VIKUS

757

harvested. Such centrifugates were used on certain occasions since they were more likely to be contaminated by sedimentable host component,s and the elementary bodies were less uniformly dispersed than in preparations obtained by t#he adsorption-elution technique. Treatment of Elementary Body Suspensions with Ether The ether treatment was carried out initially according to the technique described by Hoyle (1952). However, in order to achieve more reproducible results various modifications were introduced which are described in the experimental section. The general techniques were as follows : (a). Ether-treated elementary bodies (EEB). Squibb’s anesthetic ether was added to the EB suspensions in the desired concentration and the mixtures were incubated at given temperatures for varying periods of time with frequent manual or continuous mechanical agitation on a magnetic stirrer. Fresh ether was added as required to replace any loss due to evaporation. The aqueous and ether phases were separated by means of a separatory funnel. Excess ether was removed from the aqueous phase initially by incubation at 37” overnight but later by gently bubbling nitrogen through the suspension. This type of preparation is referred to as EEB. (b). Ether extract (EE). The ether phase was evaporated down to a small volume (1 or 2 ml) by means of a desiccator or with bubbling nitrogen and then squirted with a fine capillary pipette into warm buffered saline solution. The residual ether in the resulting opalescent, mixture was removed by evaporation. The final preparation (EE) was clarified by low-speed centrifugation, when necessary. (c). Separation of the hemagglutinin (HAP) and soluble antigen (SF) fractions. EEB suspensions, prepared as described, were chilled in an ice bath and absorbed with 10% packed chicken red cells for 1 hour at 0”. The red cells with the adsorbed hemagglutinins were removed by centrifugation in the cold, washed once with ice-cold BSS and resuspended to the original volume in phosphate buffered saline containing 1% RDE and antibiotics. Elution was allowed to take place in a 37” water bat,h for 234 hours. After removal of the red cells by centrifugation, the eluate constituted the hemagglutinin fraction (HAF). The supernatant fluid after adsorption of the EEB suspension with red cells was again adsorbed with 5 to 10% red cells to insure complete removal of the hemagglutinins. The cells of the second adsorptioll were

758

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S. LIEF

AND

W.

HENLE

washed and added to those used for preparation of the hemagglutinin fraction. The twice adsorbed supernatant, fluid comprised the soluble antigen fraction (SF). Infectivity

Titrations

The technique used has been fully described (Henle, 1949). Hemagglutination

Tests

Hemagglutinin titrations were performed in duplicate using 1% suspensions of chicken and guinea pig red cells, respectively, and, on occasion, in triplicate employing also human type 0 erythrocytes. Since guinea pig and human red cells always gave identical results, only tests with the former are recorded in the text. Serial two-fold dilutions were made in Verona1 buffered saline solution and to 0.4 ml of each dilution 0.2 ml of red cell suspension was added. The mixtures were incubated in the cold room and readings were made according to the patterns of the sedimented cells. The last dilution of a preparation giving distinct though partial agglutination was taken as the endpoint. Inasmuch as sedimentation of mammalian cells did not occur before 2 or more hours, it was found most practical as a standard procedure to read the tests after overnight incubation. With RDE-treated materials, titrations were carried out in 2 % sodium citrate saline or the test was performed as usual and read in the cold. Complement Fixation

Tests

Antisera were used which had been obtained from guinea pigs by methods to be described in detail elsewhere (Lief, in preparation). All sera were inactivated at 56” for 30 minutes and then absorbed with sheep erythrocytes. For detection of soluble antigen (S), the pooled sera of animals were used which had received active L347 virus intranasally plus an intraperitoneal booster dose of S antigen. The anti-S titer of this serum was 1: 128 while the homologous anti-V titer was < 1:32. It did not react with the V antigens of any of the other strains studied. For assay of the virus antigen (V), sera produced by intraperitoneal immunization with ultraviolet-inactivated virus of the various strains were employed. All had high titers of homologous anti-V and minimal or no detectable titers of anti-S. The anti-PR8 V serum was used unabsorbed or absorbed with Melbourne virus to differentiate between cross-reacting and strainspecific V components of PR8 virus. The anti-Melbourne serum was

SOLUBLE

ANTIGEN

IN

INFLUENZA

VIRUS

759

absorbed with L,47 (removal of anti-S) to detect cross-reacting V antigen present in PR8 preparations as well as for assay of Melbourne V. None of these sera reacted with uninfected chick embryo materials. In order to discover whether host materials were concentrated in any single fraction following ether treatment, sera were used which had been ohtamed from guinea pigs or rabbits immunized intraperitoneally with normal allantoic fluid or membrane. Sera from normal animals were employed as controls. For the complement fixation test multiple sets of serial two-fold dilut’ions of the various viral preparations were made in veronal buffered saline solution using 0.1 ml amounts per tube. Each set received then 0.2 ml of an equal mixture of guinea pig complement (approximately 2 units) and one of the sera in appropriate dilution. The optimal serum dilution to hc employed was predetermined by block titrations; i.e., by t)itrating the serum against decreasing dilutions of standard antigens. After incubat,ion at 4” overnight, 0.2 ml of sensitized sheep red cells (1%) was added to each tube and the test was incubated further at 37” for one hour. Readings were made either at this time or after the cells had settled on standing in the cold room. The last dilution of a virus preparation giving 3 or -4+ fixatjion was taken as the endpoint EXPERIMENTAL

Initial experiments were carried out following in all essential details the technique described by Hoyle (1952), with these exceptions: (a) the PR8 instead of the DSP (t,ype A, 1943) strain was employed; (b) guinea pig antisera were used instead of human convalescent or ferret immune sera; (c) hemagglutination tests were performed with chicken (CHA) and human red cells as well as with guinea pig erythrocytes (GPHA); and (d) mainly elementary body suspensions (EB) free of direct,ly detectable 8 antigen were studied. The results obtained were not readily reproducible in every aspect, as was also noted by Hoyle et al. (1954). In various experiments with the PR8 strain the GPHA titers were found to increase on ether treatment, to remain stationary, or to decrease. The CHA levels were always reduced but to variable extents. The losses in HA activity were accompanied by decreases in V antigen levels in some tests but not in others. Occasionally the EB suspensions possessed S activity prior to exposure to ether, as in Hoyle’s studies with the DSP strain. Correlation of the amount. of S released by ether from initially S-free EB suspensions t,o the CHA tit,ers

760

F.

S. LIEF

AND

W.

HENLE

of the original EB preparations revealed considerable variations. All the preparations reacted to some extent with antiserum against host components. The ether phase material (EE) fixed complement in the presence of all sera including the normal control serum. Variability of the results indicated that standardization of the method was essential in order to obtain reproducible results. It appeared to be particularly important to control the agitation of the virus-ether mixtures, since losses in various activities seemed to be dependent upon the vigor of manual shaking. Standardization

of Ether Treatment

All experiments on standardization were performed with the PR8 strain. The following modifications in general techniques were introduced : (a) 1% RDE was employed during all elution processes in order to increase the recovery of virus from the red cells and to shorten the time of experimentation. It was found that in the presence of RDE, 2 to 235 hours at 37” provided nearly 100% recovery. (b) Penicillin (500 units per milliliter) and streptomycin (100 pg/ml) were added to all eluting mixtures. (c) Ether and virus mixtures were kept continuously agitated by means of magnetic stirrers to provide uniform exposure of all virus particles to the solvent. The rheostats were set at a point just sufficient to ensure thorough mixing, and for given containers and volumes of liquid, a constant setting was always used. (d) Ether and aqueous phases were separated in separatory funnels with care so as not to disturb the interface; and (e) residual ether was evaporated by bubbling nitrogen through the ether-containing materials instead of by exposure to 37” overnight. With these adjustments, further experiments were devised in order to determine the effects of (a) temperature and time of exposure to ether; (b) the relative concentrations of ether; and (c) variation in the number of virus particles in given suspensions. a. The effect of temperature and of the time of exposure. Three aliquots of an elementary body suspension containing 10,240 CBA units per milliliter were treated with 35 volume of Squibb’s anesthetic ether for varying periods of time (a) in the cold room (4”), (b) at room temperature (ZOO), and (c) at 37”. In the latter instance, the flask containing the ether-virus mixture was immersed in a 37” water bath, which in turn was set on the magnetic stirrer base. The rheostats of all three magnetic stirrers were set up at the same speed immediately upon addition of the ether. Samples were withdrawn from each flask at $g, 36, 1, 2, and 3 hours after commencing treatment. A portion of each ether-treated elementary body

SOLUBLE

ANTIGEN

IN

INFLUE1VZ.I

761

VIRGS

suspension (EEB) was absorbed with chicken red cells and the absorbed fluid used as the soluble antigen fraction (SF). The remainder of the EEB suspensions was taken to study the effects of ether on the V and hemagglutinating activities. The results are presented graphically in Fig. 1. The data suggested that room temperature was most satisfactory, maximal yields of S being obtained in about 2 hours without significant loss in V activity. The CHA titers, on the other hand, were significantly reduced by et,her at all temperatures. b. The e.fect of various concentrations of ether. An elementary body suspension containing 10,240 CHA units per milliliter was divided into

1 5

320

-

I 2 E \ u) .z : b

I

I

I

I

c

320--s=: I60

-y

M--o

80 40-

0 Ether 0 Efher V Ether

treatment treotmeot treatment freatment

01 37’15 oi R I?.T7T of 4-C

I

I

I

I

I

I5

30

60

120

18(

Period

of ether

treatment

(minutes)

FIG. 1. The effects of various temperatures and times of exposure on the results of treatment of PR8 standard virus with ether (10,240 CHA units per milliliter).

762

F. S. LIEF

AND W. HENLE

four equal parts. To three of the aliquots were added >i, $4, and 1 volume of Squibb’s anesthetic ether, respectively, and to the fourth, $5 volume of reagent ether. Treatment proceeded at room temperature and samples were taken from each of the flasks after l-, 5, lo-, and 15-minute intervals as well as after 1,2, and 3 hours. The ether-treated elementary body suspensions were processed as in the preceding experiment. The results are given in Fig. 2. From these it was evident that exposure to anesthetic ether in a concentration of 55 volume for 1 to 2 hours at room temperature provided optimal conditions. Such treatment yielded

s

40960

8

0

20460 2 10240 \ E 5120 ;

2560 : 1260 640 L-

A .E Lo Tg

160 00

% .

40

\

FIG. 2. The effect milliliter) to various room temperature.

of exposure of PR8 standard virus (10,240 CHA units per concentrations of anesthet.ic ether and of reagent ether at

SOLUBLE

ANTIGEN

IN

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763

VIRUS

- I0240

I

II

I

I

,

60

120

I5

60

120

Period

FIG. 3. The effect of varying results

of treatment

with

of ether

treatment

(mmutes)

the concentration of PR8 standard virus 35 volume anesthetic et,her at room temperaturr

on the

maximal release of S, minimal damage to the V antigen and CHA and optimal enhancement of GPHA activit’y. Reagent ether was found to be ultimately deleterious to all activities. c. The in$Yuence of variations in the concentration of elemer&wy bodies. Samples of concentrated elementary body preparations were diluted to yield suspensions containing 40,960, 10,240, 5120, 640, and 320 CHA units per milliliter, respectively. To each dilution was added ,!,,i volume of anesthetic ether and the mixtures were agitated on magnetic stirrers at room temperature for 15 minutes and for 2 hours. The results are presented in Fig. 3. It is readily seen that t,he S titers obtained after treatment corresponded to the concentration of virus in the suspensions before exposure to ether, in that about 64 CHA units released 1 unit of S in all instances where the antigen reached detectable levels. The V antigen values, likewise, corresponded to the dilution factors employed in making the ER suspensions. For least damage to the hemagglutinins, on the other hand, not only the t’emperature and time of c~xposu~~c WY~

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important factors, but also the relative concentration of ether to the number of virus particles was important. Suspensions containing 5120 HA units per milliliter or more provided best results. The Reproducibility

of Results with the Standard Treatment

Technique of Ether

In the course of these and subsequent studies numerous standard PB8 elementary body suspensions of similar hemagglutinin titers were treated at room temperature for 1 to 2 hours with 1 part ether per 2 parts of virus suspension. The elementary bodies were prepared by one or two cycles of adsorption onto and elution from red cells (EB1 or EB2). The S fraction was prepared regularly and in nearly all cases the HA fraction as well. No attempt was made to analyze further the ether phase (EE) because of its partly nonspecific activity in complement fixation tests as shown by its interaction with normal guinea pig sera. Table 1 summarizes typical results selected from seven experiments with EB1 and 8 with EB2 preparations. The IDso/HA ratios of the original virus materials ranged from 106.0 to 106.6 in the preparations tested. It is evident that in all instances on exposure to ether the CHA levels decreased about twofold, whereas the GPHA titers rose four- to eightfold. As a rule, 16-32 CHA units of the EB suspensions corresponded to one V antigen unit (CHA/V). In two of the experiments not listed this ratio was slightly higher (64), possibly on account of inaccuracies of the HA test, since in these instances also relatively less S was released (see below). The V activity was not affected by ether and all the antigen was recovered in the HA fraction. With one single exception (not shown in the table) the EB1 suspensions and all EB, preparations failed to react with anti-S. On their exposure to ether between 160 and 320 S units were released and these were largely retained in the S fraction. However, slight S activity was found in nearly all the final HA preparations. It is evident that the yield of S from standard virus was fairly uniform in that from 32 to 64 CHA units of the EB suspensions were required in order to release one unit of S antigen, the mean CHA/S ratio of all 15 experiments being 40. If one were to include the S remaining in the HA fraction the ratio would be slightly lower. Second exposure of the hemagglutinins to ether did not liberate additional S and even the antigen present before the second treatment was often not fully recovered.

SOLUBLE

IN INFLUENZA

ANTIGEN

TABLE REPRODUCIBILITY ELEMENTARY Experimerit number

1

2

3

4

___. 5

1

OF RESULTS OBTAINED BODIES TO ETHER UNDER

Hemagglutinin

units/ml

ON EXPOSURE OF PRR STANDARD CONDITIONS

Complement fixing antigens units/ml

CHA/V


I

32

320 320 320

1

5120 40960 40960 <5 ~-___-___

320 320 320
(10 240 20 240 .~-_--~

2

5120 81920 40960

320 320 320

CHAa

GPHAa

Vb

EB, EEB SF

10240 5120 <5

10240 40960 <5

320 320
EB, EEB HAF SF __

10240 5120 5120 <5

10240 40960 40960 (5

EBI EEB HAF SF

10240 5120 5120 <5

EBz EEB HAF SF --

10240 5120 5120

EB? EEB HAF SF

10240 5120 5120 <5

EB2 EEB HAF SF

F--

Ratios

~--GPHA CHA/

Preparation

<20 240 30

?ir,

32 64

32 4X

!4 ?6 ~-

-

2

32 43

Y6 16

240

5120 40960 40960 5

640 640 640 <20

<20 320 so 320

2

10240 5120

5120 40960

320 320

<20 320

2

5120 <5

81920 <5

320 <20

40 240

<5

CHA:S

32

36 !4

<20

<5

765

VIRUS

16 >*R’ ?4

32

___.~ 6

a CHA and GPHA cells, respectively. b V and 8 = virus

= hemagglutinin and soluble

antigen,

levels

with

32 16

chicken

32

?i,j and guinea

pig red

respectively.

The h’fect of Ether on Various A Strains Other than PR8 Elementary body suspensions were prepared from 20-hour harvests of allantoic fluids of eggs injected with the Melbourne, L347, and Philadelphia 1951 strains, respectively. It was noted that the 2 A prime strains

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differed from the older PR8 and Melbourne viruses in that after one cycle of adsorption onto and elution from chicken red cells the EB1 preparations always reacted with anti-S. As will be described in detail in the succeeding paper (Lief and Henle, 1956a), this external S could be removed readily by second adsorption-elution cycles. For this reason, EBz preparations of these strains were employed in order to study the release of S from within the virus by ether. Each virus preparation was treated with ether in the standard manner and the soluble antigen and hemagglutinin fractions were separated. For the assay of soluble antigen, the anti-S serum produced by immunization with the L347 virus, was diluted sufficiently so that no anti-V was present which would have confused the results with the L347 preparations. For the assay of V antigens, antisera to the homologous strain were employed which were devoid of anti-S activity. The results of representative experiments carried over a 2-hour period are presented in Table 2. As can be seen, the CHA activity of the Melbourne strain increased on ether treatment and this effect was confirmed in several additional experiments. The L347 and Philadelphia 51 strains, on the other hand, TABLE

2

RESULTS OF EXPOSURE OF ELEMENTARY BODIES OF DIFFERENT STRAINS A VIRUS TO ETHER UNDER STANDARD CONDITIONS OF INFLUENZA

Strain

Melbourne

La47

-_ Philadelphia 51

a Measured

Prepara-

Hemagglutinin units/ml

Complement fixing antvpxs units/ml Va

S

2560 40960 40960 <5

320 320 160 (20

<20 240 <20 240

2560 1280 1280 <5

5120 10240 10240 <5

160 160 80 <20

<20 160 10 160

10240 5120 5120 <5

10240 40960 20480 5

160 160 160 <20

<20 240 10 240

tmn

CHA

EBI EEB HAF SF

5120 20480 20480 <5

EBI EEB HAF SF EBz EEB HAF SF

by homologous

GPHA

anti-v

sera.

Ratios :%

CHA/V

2

CHA/S

16 w 35

w 36 56

1

21.5

16 16

64 % .+c

43

SOLUBLE

ANTIGEN

IN

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767

VIHITR

behaved more like RR8 virus in that losses in the CHA titers were regularly noted, which in other tests particularly with L&7 were on occasion more pronounced (see Table 3). The GPHA levels rose in all cases presented. However, with L847 this increase was somewhat less pronounced and in another test even a loss was measured. Although some of these variations may be related to the concentrat,ion of virus employed (see above) it would appear that the L,47 strain is possibly more susceptible to detrimental effects of ether. The V antigens were not affectled as long as there was no marked destruction of the HA act#ivities. In each instance considerable quantities of soluble antigen were released which were largely recovered in the S fractions and only small amounts were retained in the HA preparations. Although the CHA/V and CHA/S ratios obtained in the Melbourne and La47 series recorded in the t,ahlt were somewhat lower than those seen with t’he PR8 strain, other expcriments gave higher values and thus this difference appears t,o be without, significance.

a. Infectivity. The release of S from preparations of elementary bodies is accompanied by loss of infectivity (Hoyle, 19X2). As shown in Table 3, with all the strains studied the infectivity decreased by 8 to 9 log,, unit’s in the first 15 minutes of exposure to ether; i.e., during the period in which most of t)he soluble antigen is liberated. However, it has not been possible TABLE P:FFECT

OF

EXPOSURE

OF

ELEMENTARY

3 BODIES Period

Strain

0

Test

TO

ETHER

of exposure 15

to ether hrl

ON

INFECTIVITY

(minutes) 120

180

PR8

IIMmI CHA/ml

(lee)

9.9 5120

1.5 2560

1.5 2560

0.8 1280

0.6 1280

&felbounrc~

ID&,/ml CHA/ml

(log)

10.0 10240

1.7 10240

1.1 80480

0.7 20480

1.1 20480

1,347

ID,Jml CHA/ml

(lop)

10240

1.5 1280

0.5 1280


0.8 1280

ID,,/ml CHA/ml

(log)

9.0 3840

0.5 480




Philadelphia

51

9.7

610

768

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AND

W.

HENLE

to render the preparations regularly noninfectious by prolongation of the ether treatment for as long as 3 hours. It was thought that some viable virus particles might have splashed onto the wall of the flasks above the levels of the ether-virus mixtures when the magnetic stirrers were put into motion, which in the process of removal of the mixtures after ether treatment could then contaminate the aqueous phases. However, careful removal of the aqueous phases did not alter the results. It is also possible that the EB preparations contain aggregates and that the virus particles in the interior are shielded from the effect of ether. This point remains to be clarified. b. Sedimentation by high-speed centrifugation. Elementary body suspensions of standard PR8 virus were treated with ether for varying periods of time and then centrifuged in the Spinco preparative centrifuge to discover whether any changes in sedimentation were discernible. A portion of each material treated for different lengths of time was centrifuged at 25,000 rpm for 30 minutes and another portion was spun at 30,000 rpm for 60 minutes. The upper halves of the supernatant fluids were carefully removed by Pasteur pipettes and examined for hemagglutination together with the resuspended sediments. It can be seen from Table 4 that with prolongation of exposure to ether the hemagglutinins sedimented with increasing difficulty even at 30,000 rpm for 1 hour. Whereas in untreated material only 16 % of the HA remained in the supernatant fluid, after 2 hours of ether treatment, 50% or more was not, sedimentable. Only the CHA titers are presented in the table. The results with guinea pig cells were confirmatory except that the titers were 4 to 8 times higher in the ether-treated materials than those observed with chicken cells. TABLE GANGES IN SEDIMENTATION HIGH-SPEED CENTRIFUGATION

4

OF PR8 HEMAGGLUTININS FOLLOWING EXPOSURE

(CHA) TO

BY

ETHER

Period of Exposure to Ether (Minutes) Preparation

0

15

60

120

180

Original 25000 rpm (30 minutes)

Supernate Sediment

10240 640 5120

10240 2560 2560

5120 5120 2560

5120 2560 1280

5120 2560 640

30000 rpm (60 minutes)

Supernate Sediment

640 5120

2560 1280

2560 1280

5120 640

5120 640

SOLUBLE

ANTIGEN

IN

INFLUENZA

VIRI-S

769

It cannot be said that the decrease in sedimentation was due to breakdown of the virus into smaller particles since a loss in densit,y would tend to give similar result’s. DIscussIoN

Treatment of influenza virus suspensions wit’h ether leads to the release of soluble antigen and this component can subsequently be readily separated from the hemagglutinating components bearing the V ant’igen. When these experiment’s were carried out according t)o the method originally described by Hoyle (1950, 1952) the results were variable. however, and losses in HA and V activities to varying extents were not,ed. By determinations of the optimal temperature, t’ime of exposure, and concentrations of ether and elementary bodies, as well as by introduction of such technical aids as magnetic stirrers, a st#andard procedure was developed which not only gave reproducible result’s but yielded maximal quantities of S antigen with minimum damage or loss to hemagglutinins and V antigen. Elementary body suspensions of the La47 and Philadelphia 1951 strains which were prepared from infected allantoic fluids by one cycle of adsorption ontjo and elution from red cells always contained directly det’ecatahle S in agreement with Hoyle’s experience with the DSP strain. Exposure to ether of such EB1 preparations did not readily permit differentiation between removal of “external” S or release of “internal” antigen. although some increases in S titers might be discernible (Hoyle et al., 1954). As will be shown (Lief and Henle, 1956a), the external portion is separable from the hemagglutinin by a second adsorption-elution cycle wilhou2 prior ether treatment. However, EB1 suspensions of the PR8 and Melbourne strains, which rarely reacted per se wit,h anti-S, and EB2 preparations of all strains, which were regularly free of direct,ly detectable S, on exposure to ether always yielded high titers of soluble antigen. Thus it appears that considerable quantities of S are located within the confines of the virus particle. The internal position of S is suggested by the facts that (a) uniform amounts of antigen are liberated by ether from standard (infectious) virus in that on the average about 40 CHA units of EB suspension yield 1 unit of S ; (b) incomplete virus, as will be shown (I,ief and Henle, 19566), releases less S; and (c) the release of S is accompanied by nearly complete loss of infectivity. Hoyle (1950, 1952) suggested that ether caused the disintegratioll of elementary bodies by dissolution of a lipid membrane which en(*loses

770

F.

S. LIEF

Ai\iD

W.

HENLE

smaller hemagglutinating and S antigen particles. This hypothesis appeared to be supported by the fact that the hemagglutination titers increased as a result of ether treatment as measured with guinea pig red cells. However, as shown above, the rise in GPHA was accompanied in three of the four strains studied by a decrease in CHA levels so that the CHA/GPHA ratio fell eight- to sixteenfold. Even with the fourth strain (Melbourne), which showed increased CHA as well as GPHA activities, the changes were disproportionate so that the CHA/GPHA ratio also was reduced at least fourfold. Furthermore, the V antigen which is associated with the hemagglutinating components remained largely intact in contrast to Hoyle’s findings which revealed nearly complete loss. These results do not necessarily support the suggested emergence of numerous smaller HA components since in that case a rise in V activity should be expected unless such an effect were counteracted unit for unit by inactivation of V antigen by ether. Although decreases in V activity were often noted in the present studies prior to standardization of the ether technique, such losses were observed, as a rule, only when the CHA as well as the GPHA activities were also markedly affected. The constancy of V activity in t’he standardized procedure suggests that the elementary body remains an entity. The data then could indicate that ether treatment alters the surface of the virus particle in such a fashion as to reduce receptor sites for chicken red cells and to increase receptor sites for guinea pig or human erythrocytes. In this process the infective property is lost and soluble antigen escapes. As a result of the loss of S the particle may lose in density so that it is less readily sedimentable by gravitational forces. Although this interpretation would fit the experimental results recorded above, it does not tally with the electron micrographs of ether-treated virus suspensions presented by Hoyle et al. (1953), which revealed the presence of many small particles varying in size from 12 to 50 rnp in diameter. Similar studies by SchSifer and Zillig (1954) with fowl plague virus also indicated disruption of elementary bodies by ether. It is possible that the standard method developed here is milder than the technique used by Hoyle and that the virus particles may be ruptured by more severe treatment. The loss of V activity reported by Hoyle (1950, 1952) may be indicative of the severity of his method. REFERENCES W. F. (1943). The immunological response to influenza virus infection as measured by the complement fixation test. J. Exptl. Med. 78, 347-366. FRIEDEWALD, W. F. (1944). Quantitative differences in the antigenic composition of influenza A virus strains. J. Exptl. Med. 79, 633-647.

FRIEDEWALD,

SOLUBLE

.tSTIC;EX

Ipu’ IXFLUENZ1

VIRGS

ii1

FULTOS, F. (1949). Growth cycle of influenza virus. Nature 164, 189-190. HENLE, W. (1949). Studies on host-virus interactions in the chick embryo-in-

fluenza virus system. I. Adsorption and recovery of seed virus. J. Exptl. Med. 90, l-11. HENLE, W., and HENLE, G. (1949). Studies on host-virus interactions in the chick embryo-influenza virus system. III. Development of infectivity, hemagglutination and complement fixation act,ivities during the first, infectious cycle. .I. Exptl. Med. SO, 23-37. HENLE, W., and WIENER, M. (1944). Complement fixation antigens of influenza viruses type A and B. Proc. Sm. Exptl. Biol. Med. 67, 176-179. HENLE W., HENLE, G., GROUPIE, V., and CHAMBERS, L. A. (1944). Studies on complement fixation with the viruses of influenza. J. ZmmunoE. 48, 163-180. HOYLE, L. (1945). An analysis of the complement-fixation react’ion in influenza. J. Hyg. 44, 170-175. HOYLE, L. (1948). The growth cycle of influenza virus A. 4 study of the relations between virus, soluble antigen and host cell in fertile eggs inoculated with influenza virus. Brit. J. Exptl. Pathol. 29, 390-399. HOYLE, L. (1950). The multiplication of influenza virus in the fertile egg. .I. H!/g. 48, 277-297. HOYLE, L. (1952). Structure of the influenza virus. The relation between biologiral activity and chemical structure of virus fractions. J. Hyg. 60. 229-245. HOYLE, L. and FRISCH-NIGGEMEYER, W. (1955). The disintegration of influenza virus particles on entry inbo the host cell. Studies wit,h virus labeled with radiophosphorus. J. Hyg. 63, 474486. HOYLE, L., JOLLES, B., and MITCHELL, R. G. (1954). The incorporation of radioactive phosphorus in the influenza virus and its distribution in serologically active virus fractions. J. Hyg. 62, 119-127. HOYLE, L., REED, R., and ASTBURY, W. T. (1953). Electron microscope st,udielr of the structure of the influenza virus. Nature 171, 256. KIRBER, M. W., and HENLE, W. (1950). A comparison of influenza complement fixation antigens derived from allantoic fluids and membranes. J. Zmnmol. 66. 229-244. LENNETTE, E. H., and HORSFALL, F. L. Jr. (1941). Stlldies on influenza virus. The complement-fixing antigen of influenza A and swine influenza viruses. .I. Exptl. Med. 73, 581-599. LIEF, F. S., and HENLE, W. (1956a). Studies on the soluble antigen of influenza virus. II. .4 comparison of the effects of sonic vibrat.ion and ether treat,ment of elementary bodies. Virology 2,772-781. 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. SCHBFER, W., and ZILLIG, W. (1954). ifiber den Sufbau des Virus-Elementarteilchcns der klassischen Gefltigelpest. I. Gewinnung, physikalisch-chemische und biologische Eigenschaften einiger Spaltprodukte. 2. Naturforsch. 9b, 779-788. WIENER, M., HENLE, W., and HENLE, G. (1946). Studies on the complement fixation antigens of influenza viruses types 4 and B. .T. Exptl. Med. 83, 259-279.