Studies on the soluble antigen of influenza virus

VIlXOLOGY 2, 7%%-810 (19%)

Studies

on the Soluble

Antigen

of Influenza

Virus

IV. Fractionation of Elementary Bodies Labeled with Radioactive Phosphor& KURT PAUCKER,~ FLORENCE S. LIEF, AND WERNER

HENLE

Division 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 extent of incorporation of radioactive phosphorus into infectious and incomplete influenza virus particles. It was found that regardless of the IDco/HA ratios of the progenies, the CPM/HA ratios were of the same order; i.e., noninfectious hemagglutinins were as radioactive as infectious virus. Following treatment of labeled st’andard elementary bodies (EB) with ether, 15 to 20% of the isotope appeared in the ether phase (EE). On separating the aqueous phase (EEB) into HA and S antigen fractions, it was noted that most of the remaining Pa2 was associated with the latter. Chemical fractionation of labeled standard EB suspensions by a modification of the Schmitt-Thannhauser method confirmed previous reports that the isotope was mainly in the phospholipid (hot alcohol soluble) and in the nucleic acid (hot TCA soluble) fractions. The EEB preparations revealed a relative increase of radioactivity in the cold TCA fraction and decreases in the alcohol and hot TCA extracts. Thus exposure to ether resulted in the breakdown of some viral constituents. Most of the residual phospholipids (not removed by ether treatment) were found to be associated with the HA particles, whereas the labeled components soluble in hot TCA were largely recovered in the S fractions. On chemical fractionation of labeled incomplete virus preparations, it was noted that the relative concentrations of the isotope in the hot TCA extracts decreased with the reduction of the ID,o/HA ratios of the progenies. Corresponding increases were noted in the cold TCA fractions. However, t,he ratios between the CPM values of the hot TCA extracts and the S antigen units present in the virus were found to be of the same order regardless of the composition of the progenies studied. i The work described in this paper has been supported by a grant-in-aid the National Institutes of Health, United States Public Health Service. 2 Postdoctorate Fellow, United States Public Health Service. 798

from

FRACTIONATION

OF

I’““-LABELEI)

VIRUS

79!)

Various difficulties were encountered in these studies, which are discussed together with the significance of the results and their relation to previous biological, chemical, and physical observations. INTRODUCTIOK

It was found (Lief and Henle, 1956c) that the formation of incomplctr virus in the allantois of chick embryos, as obt,ained under certain conditions of infection wit,h influenza virus, is associated with a diminished incorporation of soluble (S) antigen. Wit’h successive ten-fold decreases in the ID,,/Ha ratios of the progenies, which reflect the degree of incomplete virus formation, the amount of S released by exposure of the eltamentary bodies t,o ether decreased concurrent’ly in approximat,ely twofold steps. This rate of reduction of incorporation of S antigen resembled the lowering of ribose nucleic acid cont’ent, observed under similar conditions by Ada and Perry (1955). The similarity rnay be more thall incidental. Present evidence indicates that the S antigen represents a ribose nucleoprotein (Hoyle, 1952; Ada and Perry, 1954; Sch&fer and Zillig, 1954). It was hoped that additional information ou the nature of S ant,igen could be obtained by (a) labeling of standard and incomplete virus preparations with radioa&-e phosphorus (P) ; (b) separating the HA and S components following exposure of the labeled elementary bodies t’o et,her; and (c) fractionating of the various preparations by a modificat~ion of the Schmitt-Thannhauxer t,echnique. The preliminary results of these experiments have been reported (Henle, 1956). Isotope t’echniclues were employed also by Hoyle ci nl. (I954) in an approach to tht> study of the HA and S components. MATERIALS

ASI>

RIETHOIM

All csseutial techniques for the production of elementary body suspeusions, the treatment! of the virus mat)erials by et)her and the methods employed for the biological assays have been described or referred to in t)he preceding papers of this series (Lief and Henle, 1956a, b, c). Pro~l~ction of labeled z$rus. The met’hods used have been described in detail previously (Liu et al., 1954).” Briefly, adequate numbers of 9 to loday old rhick embryos were injected allantoirally with 120-130 PC: of 1’32per egg. After au interval of 38-72 hours the eggs were inoculat,ed wit’h various types of seeds and the alluntoir fluids were collected aft,el further incubation at :37’ for 20-24 hours. Thrl virus was adsorbed itI the 3 The advice

and aid of I)r. Oscar C’. I,iu is gratefully

acknowledged

800

K.

PAUCKER,

F.

S. LIEF,

24ND

W.

HENLE

cold onto chicken red cells (1% final concentration). The cells were removed by centrifugation, washed four times in ice-cold saline and then resuspended in sufficient amounts of 0.01 M phosphate buffered saline solution (BSS), containing RDE, to affect a two- to four-fold concentration of virus over that in the original allantoic fluids. Elution was permitted to take place at 37” for 255 hours and the separated eluate was dialyzed overnight against 20 volumes of BSS. The adsorption-elution procedure and dialysis were repeated a second time in the same fashion. The second dialyzed eluates, which represented about an eight-fold reduction in volume as compared to the original volume of infected allantoic fluid, served as the suspensions of P32-labeled elementary bodies. Chemical fractionation. For these studies the Schmitt-Thannhauser technique was used as modified by Graham (1950) and described in detail previously (Liu et al., 1954). However, since the labeled virus preparations often still contained traces of free P32,it was deemed advisable to remove these with cold t,richloroacetic acid prior to extraction with hot alcohol. This change in procedure resulted in a relative distribution of the isotope somewhat different from that previously reported (Liu et al., 1954), as will be shown in the text. Assay of radioactivity. The techniques employed have been fully described (Liu et al., 1954). EXPERIMENTAL

The Incorporation

of P32 into Incomplete Virus

A number of experiments were carried out to compare the incorporation of P32into infectious and noninfectious elementary bodies. Some of the results are summarized in Table 1. As can be seen, the degree of incorporation of P32did not differ significantly in the individual experiments, regardless of whether the progenies corresponded to standard virus with ID,,/HA ratios of > lo6 or contained high proportions of incomplete virus as evident from ratios as low as lo*. Furthermore, there was no apparent difference, whether undiluted passage or heated standard virus were employed for production of incomplete virus. All labeled virus suspensions were absorbed with chicken red cells in order to test for free Ps2. The percentage of HA absorbed exceeded only slightly that of adsorption of radioactivity. Thus, at most a small proportion of the isotope was present in a free state. Elementary body suspensions were prepared under similar conditions also from nonradioactive embryos. When these were incubated in vitro with either free P3z

FRACTIOXATIOK

OF

P32-LA13ELl!X)

TABLE INCORPORATION

OF P32 INTO

801

VIKIX

1 IXCOMPLWE

VIRUS

WO-130 &/egg) Progenies

Inoculum Dilution

IDso/HA

CPM/HA

ST A ST 2 days 37°C A ST 4 days 37°C

1o-- i

lo”,’ lo”-” 102.”

0.28 0 .3R 0.84

ST A ST 1 day 37°C A ST 2 days 37°C A ST 3 days 37°C

100 10” 100 100

106.2

103.9

0.37 0.36 0.55 0.50

106.2 lo”,’ 103,’ 101.6

0.68 0.75 0.48 0.45

Seed

10” 10”

ST

lo- 6

UPY UP3 UPa

100 100 10”

lOS.fi 101.”

or with labeled normal allantoic membrane suspensions for periods up to 24 hours at 37”, the virus particles failed to acquire radioactivity. The results agreed in every aspect with those previously reported on incorporation of P35 into noninfectious hemagglut.inins obtained on infection of HeJ,a cells (Henle et al., 1955). Distribution

of P3? among Fractions of Ether-Treated Virus

Attempts were made to determine the distribution of the isotope among various fractions obtained on exposure of labeled standard and incomplete elementary body (EB) suspensions to ether by t’he usual technique (Lief and Henle, 1956a). The aqueous (EEB) and ether phases (EE) were separat’ed and the EEB suspensions were then divided into the HA and S fractions and in one of the tests the red cells used in this process were retained after completion of the elution procedures and digested in fuming nitric acid (RBC). The assays of radioactivity (CPM/ml) are shown in Table 2. The experiments were hampered by a number of problems. (a) It was not always possible to obtain inclomplete EB suspensions of sufficient HA activity to insure minimal damage to the biological properties by ether (Lief and Henle, 1956a). (b) The EEB preparations revealed throughout decreases in radioactivity but the missing art,ivit,y was not

802

K.

PAUCKER,

F.

S. LIEF,

TABLE DISTRIBGTION

Number of CHA units exmployeda (EB)

1 2 3

Type

IDso/HA

ST ST ST

106.2 106,’ 106.3 -~106.’ 105.6 104.3

UPI ASTld

AST2d AST3d ST up* UPI UP4

-!

Vt..

HENLE

2

OF RADIOACTIVITY IN ELEMENTARY

Experiment number

AND

FRACTION BODIES

!-

OF ETHER-TREATED CPM/ml Fractions T

EB

EEB

10,240 12,800 10,240

7757 2360 7316

7265 2080 7040

40,960 40,960

3185 3550 473 242 650 3842 2893 262

103.9

7,680 3,840

4890 4840 1173 601

106.2 104.’ 103.1 104.5

2,560 10,240 10,240 1,280

1275 4880 4040 593

-

1 EE

316 345

745

261 967 894 88

-

HAF

SF

145 10176 260

3480 731 3230

-

725b 795b 52 18

2040 2085 369 182

-

45 1170b 884b 12

446 2027 1445 167

RBC

30 82 55

10

a Hemagglutinin for chicken red cells. b Incomplete separation of S; i.e., 20-80 S units in HAF.

always completely recovered in the EE fraction. While the losses were relatively insignificant in preparations of high initial virus content, they amounted to up to 40 % in those of low titer. It is likely that the missing radioactivity was present in precipitates which often formed at the interface between ether and aqueous layers. These precipitates, included in the EE preparations, made accurate sampling of this material impossible. (c) Comparison of the counts obtained with the HA and S fractions revealed that, with one exception, the former were lower than the latter by factors of 3 to 24. This variability may have several reasons. First of all, the sums of radioactivities of HAF and SF never matched those of the EEB suspension from which they were derived. The losses amounted t’o lo-50%. The HA test often failed to detect commensurate losses, since this technique is far less sensitive. Although some of the missing isotope was accounted for in the digested red cells after completion of the elution procedure (RBC), the quantities found did not balance the deficit. The RBC digests, on the other hand, formed crusty, uneven layers on the planchets, so that’ the counts mere presumably too low.

FHACTIONATION

OF

P”*-LABELEJ)

VIKrS

803

Secondly, the HA fractions were not always entirely free of 8 antigen, as previously noted (Lief and Henle, 1956a, 1956c), and bhus some of their radioactivity must have been due to labeled S antigen. When no S could be detected, the HA fractions revealed $$ or less the radioartivit? of the corresponding SF. The S preparations nevrr showed residual H,\ and V antigen. In spite of the various difficulties encountered in the above expwiments, it would seem j lstifiable to state that about’ 15-20% of the isotope of the elementary bodies appears in t,he ether phases (EE) and that the radioactivity of the S fraction is on t,he average 10 times greate! than that, of the HA preparations free of detecatable S. These results do not appear to be significantly different’ from t,hose repolted by Hoyle et al. (1954). Chemical Fractionation

of Labeled Vines Materials

A number of labeled standard EB suspensions as well as the EEB, HA, and S preparations derived therefrom, were fractionated by a modification of the Schmitt-Thannhauser technique. The cold trichloroacetic acid, hot alcohol, hot trichloroacetic soluble fractions, and the residue, labeled in that order from I to IV, were then assayed for radioactivity and the relative distribution among them was calculated. As shown in Fig. 1, the results with the EB suspensions were closely reproducible, in that on the average 11, 52, 32, and 5 % of the label was found in fractions 1, II, III, and IV, respertively. After ether treat,ment, the aqueous phaw (EEB) showed increases in the percentage of the label in the cold TC.l fracLt)ion and decreases in the alcohol and hot TCA extra&. The HA preparations in these and other experiments gave variable resulk bccause (a) t,hey often contained residual S activity (Lief and Henlr, 1956 a, c); and (b) if free of detectable S, t#heir initial radioactivit’y was usually very low and thus some of the chemical fractions derived t,hercfrom yielded counts below the limits of accuracy of the technique employed. In HA suspensions free of measurable S, most of t’ht isotope (71%) was present in the alcohol soluble fract’ion (II) and about’ 20% in the cold TCA extract, whereas the other fractions contained insignifiwnt P*. In another experiment, which was carried out prior to st,andardization of t,he ether technique, the effects of the solvent, were apparent,l> more severe, in that) most, of the V and S antigen was deskoyed. In t,hat case, t,hc distribution of the isotope was reversed, 75 % in fraction I and 18 % it) fraction II. Finally, in the S preparatiotls, from 79 to 87 % of thfk

804

K.

PAUCKER,

F.

S. LIEF,

AND

W.

EEB

HENLE

HAF

..8. rTll!L

SF

t t

1 .

.

i. L urnc

. .

l

L

,Ttmnc

i. I

. l-l

.

IImlx

FRACTIONS

The relative distribution of radioactivity in the cold TCA (I), hot alcohol (II), hot TCA (III), and residual fractions (IV) of P3z-labeled standard elementary bodies (EB), the aqueous phase of ether-treated virus (EEB), and the hemagglutinating (HAF) and soluble antigen preparations (SF) derived therefrom. FIG.

1.

label was found in the cold TCA soluble and 11 to 19 % in the hot TCA fractions, but essentially none in the alcohol extract or the residue. Similar experiments were carried out with incomplete EB preparations derived from UP and AST seeds and the fractions derived therefrom on exposure to ether. The results obtained with the EB and S materials of one experiment with UP progenies are shown in Fig. 2. The relative distribution of P32in the chemical fractions changed to some extent with increasing incompleteness of the virus progeny studied. In the EB suspensions the percentage of radioactivity increased in the alcohol extract and it decreased in the hot TCA soluble fraction. In the S preparations, most of the isotope was in the cold TCA and the remainder in the hot TCA extracts. However, with a decrease in the ID&o/HA ratios of the progenies the percentage of radioactivity in the hot TCA fractions decreased, matched by a corresponding relative increase in the cold acid extract. Efforts

to correlate

the actual

counts

(CPM/ml)

obtained

in the

various chemical fractions with the number of S antigen units per milli-

FRACTIOKATION

OF

P32-LARELF:I)

VIRUS

805

EB

FRACTIONS

y in the cold TCA (I), hot FIG. 2. The relative distribution of radioactil alcohol (II), hot TCA (III), and residual fractions (IV) of PWabeled elementaq bodies (EB) obtained by standard (ST) and serial undilut,ed passage8 (UP?, UPp, and UP4) and of the soluhle antigen preparations (SF) derived therefrom on exposure to ether. liter, which were released from the elementary bodies by ether led t)o the results shown in Table 3. The CPM/S ratios for fractions I and II (cold TCA and hot alcohol extracts, respectively) rose considerably as t.he degree of incompleteness of the virus increased, regardless of whether EB, EEB, or SF were studied. Thus, no direct relationship to tjhe 8 antigen became apparent. On the other hand, the CPM/S ratios obtained with the hot TCA fractions were nearly constant. In the EB series the ratios were of the order of I with the exception of the UP, material. In t’hat case, a value of 14 was recorded, possibly on account of incomplete separation of t)he fractions. The ratios in t’he EEB and SF series were reduced to about 1. These results suggest a relationship of P antigen to the hot TCA, or nucleic acid fraction in support of previous reports indicating that S antigen is a nucleoprot8ein (Hoyle, 1952; Ada and Perry, 1954; Sch&fer and Zillig, 1954). The difference in the CPM :‘S ratios based on the hot TCA fractions of the EB as compared to t,he EEB and SF series may denot!e (a) that t,he intact virus part’icles coatjain some compounds extrartable by hot TCA, which are not related to S antigen, and which are so altered by the ether treatment, that they maJ become soluble in cold TCA or alcohol; or (b) that ether destroys some of the S activity and that t,he virus particles actually contain more antigen t,han can he determined by the method employed. This point d+

806

K.

PAUCKER,

F.

S. LIEF,

AND

W.

HENLE

TABLE 3 CORRELATION OF RADIOACTIVITY IN CHEMICAL FRACTIONS OF EB, EEB, AND SF WITH S ANTIGEN UNITS RELEASED FROM THE VIRUS

Virus preparation

EB

EEB

Type of Pm3=Y

Units of S antigen re’eased by ether

ST UP2 UPS UP4 ST UP2 UP3

UP4 SF

ST UP? UP3 ma

(1Based on S antigen

80 2ooa 120” 5 80 200” 1200 5 80 160 80 5 units

PARTICI~ES

BY ETHER Chemical Fractions

(Cold TC!4 Soluble) CPM/ml CPM/S

66 544 539 31 249 1356 1063 130 253 1342 1140 120

0.8 2.7 4.5 6.2 3.1 6.8 8.9 26.0 3.2 8.4 14.3 24.0

(Hot Alco::l CPM/ml

618 2689 2242 314 137 1456 1142 57 2 19 9 2

Soluble) CPM/S

7.7 13.4 18.7 62.8 1.7 7.3 9.5 11.4 0.03 0.19 0.11 0.40

(Hot T&I%&.$ CPM/ml

346 854 526 70 86 224 86 6 60 170 61 5

B

4.33 4.27 4.38 14.00 1.08 1.12 0.72 1.20 0.75 1.06 0.76 1.00

in S and HA fractions.

serves further study and, especially, a search for milder means of liberating S from EB appears to be indicated. DISCUSSION

As has been shown previously (Graham and McClelland, 1950; Hoyle et al., 1954; Liu et al., 1954) influenza virus readily incorporates radioactive phosphorus when propagated in the allantois of chick embryos previously injected with the isotope. The degree of incorporation depends upon the dose of P3* employed, the interval between its administration and infection and the age of the chick embryos used. It was subsequently demonstrated that the noninfectious hemagglutinins produced on infection of cultures of HeLa cells, likewise incorporated P32 (Henle et al., 1955). In the present studies, it was shown that the incomplete virus obtained following injection of chick embryos with undiluted passage or heated standard seeds became labeled to the same extent as fully infectious virus, in that the CPM/HA ratios of the various types of progenies were of the same order and differed only within the range of the accuracies of the techniques employed. As was noted with standard elementary bodies (Graham and McClelland, 1950; Liu et al., 1954) the

FRACTIONATION

OF

P32-LABELED

VIRUS

807

incomplete forms of virus incorporated P32 only when propagated in radioactive host tissues, but they were incapable of adsorbing either the free isotope or labeled host components in vitro. These results lend further strong support to the conclusion, arrived at previously on other evidence (von Magnus, 1951, 1955; Finter et al., 1955; Paucker and Henle, 1955; Henle et al., 1956), that the noninfectious hemagglut’inins are actually produced in the cells and do not represent degraded seed virus. On fractionation of labeled elementary bodies (EB) by ether a number of difficulties were encountered which prevented accurate quantitative analyses. Part of the isotope was lost on separation of the aqueous (EEB) and ether phases (EE) and on dividing of the EEB suspensions into the hemagglutinin (HA) and soluble antigen (S) fractions. The losses were the more evident the smaller the original concentration of elementary bodies. In addition, the HA preparations were not always free of S activity (Lief and Henle, 1956a, 1956c). The results obviously would have gained in significance if the radioactivity could have been related to a stable property present in all fractions. No such reference is available at present. In spite of these various handicaps, it would seem to be evident from those experiments in which an apparently clean-cut separation of HA and S was achieved, that after ether treatment the remaining isotope was associated mainly with the S fraction, as previously shown by Hoyle et al. (1954). The low levels of radioactivity encountered in the HA preparations may be due largely to incomplete removal of phospholipids from the elementary bodies by exposure to et,her. Chemical fractionation of labeled EB suspensions of standard virus by a modification of the Schmitt-Thannhauser method yield reasonably uniform results with respect to the relative distribution of the isotope. On the average, 10% of the radioactive phosphorus was soluble in cold TCA, slightly more than half in hot alcohol, about x in hot TCA, and t,he residue accounted for the remainder. The (*old acid soluble Psa may represent’ in part isotope which was not incorporated into the virus. This is suggested by the fact that on addition of red cells to labeled elementary bodies the percentage of HA adsorbed always slightly c&xceeded adsorpt’ion of radioactivity. It was evident that, the label was mainly in the phospholipid and nucleic acid fractions as reportsed previously by Graham (1950) and Hoyle et al. (1954). The higher percentage of isotope in the alcohol soluble fractions recorded in the present study as compared to that noted by Graham and Hoyle it al. can be csplained

808

K.

PAUCKER,

F.

S. LIEF,

AND

W.

HENLE

by differences in the techniques used for production of labeled virus. In as yet unpublished experiments, it was found that when the interval between injections of P32and seed virus was long (48-72 hours, as used here), the labeling of the phospholipid portion of the progeny was greater than when the isotope and seed were injected at shorter intervals, as employed by the authors cited. On exposure to ether (EEB) the relative distribution of P32 changed considerably. The percentage of the isotope in the alcohol and hot TCA extracts decreased and that in the acid soluble fractions increased. Thus ether apparently causes some breakdown of viral components, as has been suggested by Hoyle et al. (1954). Since separated S-free HA fractions revealed only low radioactivity, the chemical fractions derived therefrom often yielded counts close to background activity and thus cannot be considered accurate. Whatever radioactivity was found was present in the acid and alcohol soluble fractions. This suggests that any phospholipids not removed by ether treatment remained associated with the HA components, The corresponding data with the S fractions, on the other hand, revealed that practically all of the radioactive components were soluble in cold or hot TCA. This would indicate that the bulk of nucleic acid was in the S fraction. The cold acid soluble materials found in both HA and S fractions presumably represent largely breakdown products of phosphorus-containing compounds obtained on treatment by ether. On comparing labeled EB preparations of increasing degrees of incompleteness, the percentage of the label in the hot TCA extracts decreased and that in the alcohol soluble fractions increased. This would appear to correlate with the reduction in ribose nucleic acid (Ada and Perry, 1955), and with the increase in lipid content (Uhler and Gard, 1954). The EEB, HA, and S fractions derived from the UP series showed essentially similar changes in the relative distribution of radioactivity as the corresponding materials obtained from standard virus, except that the percentages of P32in the hot TCA fractions of the EEB and S preparations were lower in correspondence to the results obtained with the respective EB suspensions, and those of the acid soluble fractions were in turn higher. These findings would seem to be in agreement with the suggestion that S antigens of the influenza group, including fowl plaque virus, are nucleoproteins (Hoyle, 1952; Ada and Perry, 1954; Schafer and Zillig, 1954) and that the HA components are essentially protein in nature. No data on specific activity were obtained in this series of experiments nor

FRACTIONATION

OF P3'-LABELED

VIRUS

809

have any attempts been made to identify the components in the various chemical fractions. For such studies quantities of labeled virus would be required beyond those conveniently and safely produced in this laboratory. REFERENCES ADA, G. L., and PERRY, B. T. (1954). Studies on the soluble complement fixing antigens of influenza virus. III. The nature of the antigens. Australian J. Exptl. Biol. Med. Sci. 32, 177-186. ADA, G. I~., and PERRY, B. T. (1955). Infectivity and nucleic acid content of influenza virus. Nuture 176, 209. FINTER, N. B., LIU, 0. C., and HENLE, W. (1955). Studies on host-virus interactions in the chick embryo-influenza virus system. X. An experimental analysis of the von Magnus phenomenon. J. Exptl. Med. 101, 461478. GRAHAM, A. F. (1950). The chemical analysis of purified influenza virus A (PR8 strain) containing radioactive phosphorus. Can. J. Research E28, 186-195. GRAHAM, A. F., and MCCLELLAND, L. (1950). The uptake of radioactive phosphorus by influenza virus A (PRS strain). Can. J. Research E28, 121-134. HENLE, G., GIRARDI, A., and HENLE, W. (1955). .4 non-transmissible cyt,opathogenie effect of influenza virus in tissue culture accompanied by formation of non-infectious hemagglutinins. J. Exptl. Med. 101. 25-41. HENLE, W. (1956). The use of phosphorus-32 in the study of incomplete forms of influenza virus. Trans. N. Y. Acad. Sci. 18, 255-260. HENLE W., Lrrr, 0. C., PAUCKER, K., and LIEF, F. S. (1956). Studies on the hostvirus interactions in the chick embryo-influenza virus syst,em. XIV. The relation between tissue-bound and liberated virus mat,erials under various conditions of infection. J. Exptl. Med. 103, 799-822. HOYLE, L. (1952). Structure of influenza virus. The relation between biological activity and chemical structures of virus fractions. J. Hyg. 59, 229-245. HOYLE, I,., JOLLES, B., and MITCHELL, R. G. (1954). Incorporation of radioactive phosphorus in the influenza virus and its distribution in serologicall,v active fractions. J. Hyg. 62, 119-127. LIEF, F. S., and HENLE, W. (1956~). Studies on the soluble antigen of influenza virus. I. The release of S antigen from elementary bodies by treatment with ether. Virology 2,753-771. LIEF, F. S., and HENLE, W. (19566). Studies on the soluble antigen of influenza virus. II. A comparison of the effects of sonic vibration and ether treatment. of elementary bodies. Virology 2, 772-781. LIEF, F. S., and HENLE, W. (1956c). Studies on the soluble antigen of influenza virus. III. The decreased incorporation of S antigen int.o elementary bodies of increasing incompleteness. Virology 2,782-797. LIU,~. C.? BLANK,H., SPIZIZEN, J. and HENLE, W. (1954).Theincorporation of radioactive phosphorus into influenza virus. J. Zmmunol. 73, 415-425. PAUCKER, K., and HENLE W. (1955). Studies on host-virus int,eractions in the chick embryo-influenza virus system. XII. Further analyses of yields derived from heat-inactivated standard seeds. J. Exptl. Med. 101, 493-506.

810

K.

PATJCKER,

F.

S. LIEF,

AND

W.

HENLE

SCHHFER, W., and ZILLIG, W. (1954). tfber den Aufbau des Virus-Elementarteil-

chens der klassischen Gefltigelpest. I. Gewinnung, physikalisch-chemische und biologische Eigenschaften einiger Spaltprodukte. 2. Naturforsch. 9b, 779-788. UHLER, M., and GARD, S. (1954). Lipid content of “standard” and “incomplete” influenza A virus. Nature 173, 1041. VON MAGNUS, P. (1951). Propagation of the PRS strain of influenza A virus in chick embryos. II. The formation of “incomplete” virus following inoculation of large doses of seed virus. Acta Pathol. Microbial. Stand. 28, 278-293. VON MAGNUS, P. (1955). Incomplete forms of influenza virus. Advames in Virus Research 2, 59-79.