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Studies on the structure of influenza virus
VIROLOGY
8,214O
Studies II. Ultrathin
(1959)
on the Sections
Structure
of Infectious
AKSEL BIRCH-ANDERSEN The Laboratory
of Biophysics
of Influenza and Noninfectious
Particles’
AND KURTPAUCKER~'~
and InfEuenza Department, Copenhagen, Denmark Accepted
Virus
December
State
Serum
Institute,
8, 1958
The morphology of infectious and heat-inactivated influenza virus, as well as of incomplete particles obtained under various conditions, was studied in the electron microscope. Purified preparations of elementary bodies were examined in ultrathin sections and in sprays from a volatile suspending medium. The composition of the materials under study was determined by relating infectious titers to hemagglutinin content and by assaying for the amount of internally bound soluble antigen released on treatment with ether. Standard virus in spray drop preparations consisted of spherical particles of uniform size and measuring on the average 118 rnp in diameter. Sections revealed spheres with a mean diameter of 70 rnp which contained an electrondense core limited by an inner membrane, an external coat of lower density, and a less distinct external membrane. Sprays of incomplete virus derived from serial undiluted passages (UP) and from the inoculation of heated standard seed (ASTP), contained particles of variable sizes and flatter than infectious virus, as well as large baglike structures. In addition to these, completely flattened and apparently empty ghosts were encountered with virus grown in HeLa cells and with noninfectious elementary bodies extracted from chorioallantoic membranes of standard eggs (CAM). In sectioned particles of UP, ASTP, and HeLa-grown virus, electron-dense centers were usually lacking and no internal details could be discerned. Their outer structures did not differ essentially from those of the infectious agent. Sections of preparations extracted from chorioallantoic membranes containing considerable quantities of noninfectious hemagglutinins revealed mainly masses of amorphous material. Those viral structures which could be recognized, however, resembled the other incomplete viruses described. 1 A portion of this report was presented Microbiology in Stockholm, 1958. 2 Fellow of the National Foundation for 3 Present address: Research Department, delphia, Philadelphia, Pennsylvania. 21
at the 7th Infantile The
International
Paralysis. Children’s
Hospital
Congress
of Phila-
of
22
BIRCH-ANDERSEN
Heat-inactivated standard virus, longed exposure to 37”C, possessed of the infectious agent. It is thought, from other noninfectious forms.
AND
PAUCKER
although somewhat changed due to proall the internal structural characteristics therefore to represent an entity distinct
INTRODUCTION
In the past, noninfectious forms of influenza virus obtained from mouse brain, from serial undiluted passage in eggs, from egg passage of mixtures of heat-inactivated and infectious virus and from chorioallantoic membranes have been examined in the electron microscope (Werner and Schlesinger, 1954; Voss and Wengel, 1955; Pye et al., 1956; Shiota, 1956; Hellos, 1957). In most of these studies, particles adsorbed onto lysed red cell ghosts according to a technique originally described by Dawson and Elford (1949) were observed. They have been described as flattened pleomorphic structures, somewhat larger in diameter than the infectious agent and of lesser density. The preceding study (Paucker et al., 1959) revealed that on treatment of incomplete virus units with ether (Hoyle, 1950, 1952), only small numbers of structural components identified with soluble (S) antigen activity were released. This corroborated the serological findings of Lief and Henle (1956b) and the determinations of nucleic acid content by Ada and Perry (1956). In addition, the data suggestedthat fewer than normal spherical hemagglutinating components may have been incorporated into the incomplete virus struct,ure. The dimensions of the HA subunits made it seem likely that they, rather than S antigen, were responsible for the altered appearance of the noninfectious agent. This pointed to fundamental differences in the internal organization of the two forms of virus, and these have been studied further, making use of recent advances in preparatory techniques for electron microscopy. The examination of whole particles appeared of limited value in such an investigation. On the other hand, thin sections have been used to advantage in the study of vaccinin virus (Peters, 1956; Epstein, 1958) and of bacteriophage (Birch-Andersen, 1956). This method was adapted to permit the examination of ultrathin sect,ions of purified virus materials obtained under the conditions favoring the production of maximal amounts of infectious virus (Horsfall, 1954; Finter et al., 1955) or those giving rise to incomplete forms. As a source of the latter the embryonat’ed egg, which is potentially capable of producing infectious as well as noninfectious virus, and the HeLa cell known to support an incomplete cycle of growth only (Henle et al., 1955) were
SECTIOKS
OF
ISFECTIOUS
AND
NONINFECTIOUS
PARTICLES
23
selected. Since one cannot assume a priori that the same fundamental processes underlie the production of incomplete virus by various laboratory manipulations and from different sources, morphological dissimilarities among incomplete virus populations arising from different host systems might not be unexpected. The inclusion of standard virus rendered largely noninfective by heating in vitro at 37” was motivated by the hypothesis that incomplete forms may represent originally infect,ious units which were thermally inactivated in 02’0 during the incubation period (Horsfall, 1954, 1955). MATERIALS
.4ND METHODS
Virus. The PR8 strain of influenza A was used in this study. Standard virus (ST) was obtained by inoculating allantoically 11- to 13-day-old embryonated eggs with lo3 to lo4 ID,, of seed virus. The eggs were chilled and harvested after 24 hours of incubation at 37”. Undiluted passage (UP) seed was prepared essentially by the method of von Magnus (195lb). Heat-inactivated virus @ST) was obtained by incubating standard seed in rubber-stoppered centrifuge bottles in a 37” water bath for several days. Inoculation of the undiluted partially heatinactivated preparation yielded heat,ed passage virus (ASTP) as described earlier (Paucker and Henle, 1955). Viruses derived from HeLa cells were prepared as reported previously (Paucker and Henle, 1958). In order to obtain sufficiently pure preparat,ions of HeLa virus, only hemagglutinins released into the fluid medium from destroyed cells, but not those derived from mechanical disintegration of cell debris, were used for further handling. Membrane virus (CAM) was derived from chorioallantoic membranes of eggs inoculated with 103to IO4 EID,, of standard seed. The membranes were harvested after 24 hours of incubatlion at 37” and washed repeatedly in large volumes of 41/100 phosphate-buffered saline solution (BSS) at pH 7.4 until they were free of blood. They were suspended in BSS cont’aining 5% RDE,” using 1 ml per membrane, and incubat,ed overnight in a 37” water bath. After further washing, the membranes, resuspendedin the same volume, were emulsified in chilled Servall omnimixer vesselsimmersed in ice. Cellular debris was removed by low-speed centrifugation and hemagglutinins were purified and concentrated by repeated adsorptions onto and elutions from chicken red cells. 4Receptor-destroyingenzyme; obtained from the Behring Werke, Max-burg, Germany.
24
BIRCH-ANDERSEN
AND
PAUCKER
Adsorption and elution of virus. All preparations were subjected to two or more cycles of adsorption and elution during which twenty- to fiftyfold concentration of virus was attempted. Thrice washed and packed chicken cells were added to the chilled materials to constitute a final concentration of 1 to 5%. The mixtures were incubated in the cold for 1 hour and the red cells were removed by centrifugation. After being washed once in ice-cold saline solution, they were resuspended in appropriate volumes of phosphate-buffered saline containing per milliliter approximately 50 units of RDE, 200 units of penicillin, and 200 pg of streptomycin. Elution of virus was carried out for 2 hours in a 37” water bath. The red cells were then removed by low-speed centrifugation and the supernatant fluid contained the elementary body suspension. Centrifugation. All elementary body suspensions, after having undergone preliminary purification by at least two cycles of adsorption and elution, were subjected to two or more sedimentations in the Spinco Preparat,ory L centrifuge. Standard, UP, AST, and ASTP preparations were spun for 60 minutes at 51,000 g, and HeLa-grown as well as CAM viruses were centrifuged for 180 minutes at 105,000 g. After each centrifugal run, sediments were resuspended in appropriate volumes of phosphate-buffered saline and clarified for 10 minutes at 6600 g. Assays for virus activity. Egg infectivity and hemagglutination tests were performed as described earlier (von Magnus, 1951a). The amounts of internally bound soluble antigen were determined by subjecting elementary body suspensions to treatment wit’h ether as described in the preceding report. The techniques used for complement fixation tests (Henle et al., 1956) as well as the preparation of anti-V and anti-S guinea pig sera (Lief and Henle, 1956a) have been reported. Preparation of sections. The concentrations of individual virus materials were adjusted to approximately 104.5 hemagglutinin units per milliliter. They were sedimented once more at the same speeds as those used during earlier cenOrifuga1 runs. After evacuating the supernatant fluid by gentle suction, the virus pellets were removed with a scalpel and embedded in 1.8 % Difco agar made up in phosphate-buffered saline of pH 7.4. For this purpose plastic cylinders, 2 cm in diameter and 1.5 cm in height, had been constructed through which two holes measuring 0.5 cm in diameter had been drilled. These were blocked at one end with rubber stoppers and filled to the half-way mark with agar which was permitt,ed to solidify. The virus pellets were deposited on top of the latter and the molds were then completely filled with agar cooled down
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
25
to approximately 50”. After hardening in the cold, the stoppers were removed and the agar cylinders were pushed out by means of a metal rod. Only the center section containing the virus pellet was used for further processing. Essentially, the method described by Sjijstrand (1956) was followed, except that fixation was carried out for 1 hour at room temperature in 1% osmium tetraoxide made up in phosphate-buffered saline at pH 7.4. Most of the agar surrounding the pellets was carefully peeled away before embedding small portions of the latter in prepolymerized methacrylate. A Sjiistrand ultramicrotome (Sjbstrand, 1953) was used for cutting the sections, which were floated on a 20% ethyl alcohol-80 % water mixture before being deposited on Formvar-coated grids. The sections with methacrylate left in place were examined in a Philips electron microscope, model EM 100 B. The stigmator of the compensated pole piece of the instrument was routinely adjusted so that the degree of astigmatism was of the order of 0.3-0.4 mp. Spraying of virus. This technique has been described in the preceding paper (Paucker et al., 1959). EXPERIMENTAL
Selection of Virus Preparations The suitability of the materials for study in the electron microscope was determined by infectivity and hemagglutination tests. The degree of incompleteness was expressed as IDr,o:HA ratios. Complement fixation titrations with anti-V and anti-S guinea pig sera (Lief and Henle, 1956a) were performed on samples of elementary body suspensions after concentration and purification by adsorption onto and elution from chicken red cells, but prior to sedimentation in the centrifuge. As a rule none of the purified preparations reacted with the anti-S serum prior to ether treatment (EB) whereas varying amounts of soluble antigen were released after this procedure (EEB). These values were related to the HA titers of the original concentrates and expressed in terms of hemagglutinating units required to yield 1 complement-fixing S unit on treatment with ether (HA:S ratio). Table 1 lists the pertinent data used to evaluate the composition of the materials selected. Fully infectious virus grown under standard conditions (ST) exhibited ID,,: HA and HA:S ratios typical for this material. In the order of 106.271D50were equivalent to 1 HA unit, and 75 of the latter released 1 complement-fixing S unit on exposure to the solvent. The IDS,: HA ratios of a fourth undiluted passage (UP4), a single undiluted passage of stand-
26
BIRCH-ANDERSEN
AND
TABLE THE
COWOSITION
Type of PR8 Titus
OF VIRUS
ST
Infectious
1
PREPARATIONS 1Dso:HA 10s
Composition
!
6.27
i
PAUCKER
SELECTED
FOR Titers
hlaterial tested
CFV
602,500 870,000
STUDY
per milliliter
HA
EBd EEB*
MICROSCOPIC
6,400 8,000
i
CFS
EBHA:S --
75 ---
UP4
Incomplete
4ASTP
2.70
Incomplete
2.99
.HeLa
Incomplete
i
3.65”
-I-
CAM
Incomplete
4.17
EB EEB .,----EB EEB -~-~EB EEB ----,---.--i-_-‘- EB EEB
43,100 43,100 ---~-
640 480 ---__
15,150 7,590
160 80
160 60
320 320
i
----
---11,210 7,590
10 60
---_ j ?J.D.c
1 K.D.
720
---_ 505
35” -.--
1 x.1).
4AST 94 n So increase over ID,, titer of residual b No increase over external S titer before c Kot, determined. d Elementary body. e Ether-treated elementary body.
seed. ether
treatment.
ard virus inactivated for 4 days at 37” (4ASTP) and of virus derived from chorioallantoic membranes of eggs infected with diluted seed (CAM) were all subst,antially lower. This indicated that predominantly noninfectious particles were present. In agreement with earlier data (Lief and Henle, 195610) approximately ten times more HA units of UP4 and seven times more HA units of 4ASTP than of ST were needed to free 1 soluble antigen unit. In the case of HeLa virus, no increase whatever in infectious titer over that of the residual seed inoculum was noted. Repeated cycles of purification with chicken red cells could not remove all of the external soluble antigen, but no additional amounts were released by ether. The ID50:HA ratio of 4AST indicates that most of its initial infectivity had been destroyed in the course of heating. The content of S antigen, however, was of a similar order as that found in standard virus.
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
27
Virus Sprays All virus materials were sprayed prior to sectioning in order to assess their purity and to check on the morphology of whole particles. ST virus (Fig. 1) consists of spherelike units with a mean diameter of 118 rnp. While some variation in shape can be noted, their diameters fall within a narrow range. In many instances a rimlike structure, approximately 15 rnp in thickness, can be seen surrounding the virus particles. Incomplete virus, on the other hand, by whatever method produced, represents a more heterogeneous aggregation of particles, exhibiting considerable variations in shape and size. In both UP4 virus (Fig. 2) and the lASTP preparation (Pig. 3) the particles appear somewhat flattened. Ghostlike structures with well-defined rims can also be seen. The latter predominate in both the HeLa (Fig. 4) and CAM (Fig. 5) preparations. In addition, the former contains also particles approximately 9@-100 rnp in diameter which are characteristically arranged in short chains. The heat-inactivated virus (4AST) is shown in Fig. 6, a and b. The outline of these particles is not as well defined as standard virus, and they show a tendency toward clumping. These changes are probably due to mild denaturation during the process of heating. Virus Sections The morphological differences between infectious and incomplete influenza virus are more clearly brought out in ultrathin sections. Standard virus units (Fig. 7) appear in general as spherical or ellipsoidal bodies, often somewhat compressed during the cutting process. When the plane of sectioning cuts cent.rally through a particle, a dense inner region is revealed, measuring on the average 45 rnp in diameter, which is limited by a membrane approximately 40 B in thickness. This structure is surrounded by a zone of lower density about 10 mp in thickness, which appears to be enclosed by a less clearly defined outer membrane. Averaging the long and short axes of the compressed particles, introducing thereby a negligible error (in the order of 1% in this case), t’he sectioned units of standard virus have a diameter of about 70 mp. A certain number of filaments is also present in this preparation. Their outer limiting structures resemble that of the spherical particles and their diameter is of t’he same order or slightly less in most, instances. In the interior no structural detail can be discerned except at one end where electron-dense mat,erial is condensed in an area approximat,ely the size of a free virus particle. The higher magnification (Fig. 8) shows
FIGS.
l-4
1. Standard virus. Spherical elementary bodies. 2. Incomplete virus after four serial undiluted egg passages. Particles of size and shape. 3. Incomplete virus after egg passage of heated standard seed. Note appearance of particles and some filamentous forms. 4. Incomplete virus from HeLa cells. Ghosts and small flattened spherical sometimes arranged in chains. FIG.
FIG.
variable FIG.
flattened FIG.
particles, FIGS.
shadowed.
l-4. Sprayed from ammonium Magnification: X 30,000.
acetate 28
buffer.
Palladium-platinum
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
29
FIGS. 54 a-b FIG. 5. Incomplete virus from chorioallantoic membranes producing infectious virus. Ghosts and clumped flattened spheres. FIG. 6. a and b. Heat-inactivated standard virus. Note blurred outlines of particles and tendency toward clumping. FIGS. 5 and 6. Sprayed from ammonium shadowed. Magnification: X 30,000.
acetate buffer. Palladium-platinum
that the material in the interior of the spherical standard particles is not uniformly arranged. It has a patchy appearance and it is divided into small compartments by strands of higher electron density. Sections of the UP4 preparation are shown in Fig. 9. These particles show a considerable distribution in sizes, with diameters ranging from
SECTIONS
OF
INFECTIOUS
FIG. 8. Sections of sbandard ance of centers. Magnification:
AND
virus. Note X 225,000.
NONINFECTIOUS
uneven
density
31
PARTICLES
and patched
appear-
32
BIRCH-ANDERSEN
FIGS. FIG.
varying FIG.
standard
AND
PAUCKER
9-10 a-b
9. Sections of incomplete fourth undiluted passage virus. Particles of sizes lacking electron-dense centers. Magnification: X 94,500. 10. a and b. Sections of incomplete virus obtained on passage of heated virus. Note absence of dense centers. Magnification: X 94,500.
FIGS. 11 a-c-12 FIG. 11. a-c. Sections of incomplete virus grown in HeLa cells. Aggregates of particles lacking dense centers. Magnification: X 150,000. FIG. 12. Sections of incomplete virus from chorioallantoic membranes. Amorphous material and some hollow virus structures. Magnification: X 94,500. 33
34
BIRCH-ANDERSEN
AND
PAVCKER
50 to 120 rnp in accordance with the micrographs of sprayed intact units. The particles are in general more compressed and most of them reveal little or no detectable internal structure; the over-all impression is that of hollow virus units. However, some spheres with electron-dense centers are also present. They may conceivably represent infect’ious particles inasmuch as incomplete preparations always possess a certain degree of infectivity. Whenever the plane of section is optimally located, two outer rings are discernible, which suggests that the limiting structures of this virus resemble that of the infectious agent. The balloonlike forms, on the other hand, are less clearly defined and may be in a stage of disintegration. Essentially a similar appearance is presented by 4ASTP
FIG.
centers
13. Sections and blurred
of heat-inactivated outlines. Magnification:
standard virus. X 94,500.
Note
electron-dense
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
35
(Fig. 10, a and b), except that the integrity of the virus structure was not as well preserved. Many aberrant forms, in a more or less disrupted state can be seen. Most of the smaller units with well-defined limiting membranes exhibit centers of low electron density. The outlines of HeLagrown virus (Fig. 11, a, b, and c) are somewhat blurred. However, purification of this mat,erial met with considerable difficuhy, as can be inferred from the micrograph of the sprayed preparation and the presence of external soluble antigen which could not be removed during repeated red cell adsorption and elution cycles. The particle diameters range mostly from 60 to 65 rnF and are thus slightly smaller t’han those of standard virus. The units show a tendency to form aggregates. Most, of them, like the other incomplete preparations, show no internal structure. Sectioning of the CAM preparation (Fig. 12) did not yield readily interpretable results. Most of the material seems amorphous and may consist in part of host material carried through the purification procedure. However, wherever units of virus morphology are recognizable, their centers are not filled with electron-dense material and thus they resemble incomplete rather than standard virus. Heat-inactivated virus (Fig. 13), on the other hand, differs considerably from the incomplete preparations studied. The particles, are fairly uniform in size, and their mean diameter is of a similar order as that of ST virus (71 mp). They also exhibit centers filled with a material of high density and the same internal patched appearance as that encountered in the infectious forms. The limiting coats cannot be recognized as clearly, but the hazy outlines may well be due to denaturation during prolonged exposure to 37”. DISCUSSION
The present study indicated that the internal structure of incomplete influenza virus particles, obtained by various manipulations and from two distinct hosts, differed markedly from that of the infectious form. Ultrathin sections of purified suspensionsof infective units revealed t,hat their centers were filled with an electron-dense material which showed a certain amount of organization. Sectioned incomplete particles obtained from serial undiluted passages(von Magnus, 1951b), the inoculation of heated standard seed (Paucker and Henle, 1955) and from HeLa cells (Henle et al., 1955) differed in one important aspect from standard virus. Electron-dense centers mere usually absent and t,hus no internal structural details could be discerned. Essentially the same held true for non-
36
BIRCH-ANDERSEN
AR;D
PAUCKER
infectious units derived from chorioallantoic membranes producillg infective virus (Henle, 1953; Granoff, 1955; Henle et al., 1956). However, only few virus structures could be identified with reasonable cerbainty within the amorphous mat’erial seen in sections. The examinat’ion of intact’ particles was carried out simultaneously. Whereas infectious elementary bodies a.ppeared as well-defined spherical units, incomplete forms derived from undiluted serial passages or from a partially preinactivated inoculum exhibited a greater assortment of sizes and sha.pes and a certain degree of flattening. This confirms in general the reports of others (Voss and Wengel, 1955; Shiota, 1956; Pye et al., 1956; Hellos, 1957). The differences in morphology observed were perhaps not as striking as those cited earlier, but the spraying from volatile suspending medium (Backus and Williams, 1950) may have minimized to some extent the distortions which occur during the conventional air-drying of preparations for microscopic st,udy. Whole incomplet’e particles obtained from HeLa cells or extracted from chorioallantoic membranes producing standard virus differed markedly from both the infectious and the other incomplete forms mentioned. They consisted largely of ghostlike structures and aggregates or chains of small, flat,tened units. These findings are at variance with observations from ot,her laboratories. Werner and Schlesinger (1953) found that part,icles obtained from rnembranes and allantoic fluids of eggs in which standard virus was produced were alike in appearance. Shiota (1956) described a narrower distribution of sizes, slightly smaller diameters, but an otherwise normal morphology of the virus derived from the CAM. However, the IDbo:HA rat)ios of their prepambions (lOfi,O or more) and the method of ext,ruction indicate t)hat’ not sufficient care may have been t’aken in removing infectious virus which, after migration through t’he host cell membrane, had remained superficially attached t’o it. On the other hand, the morphology of noninfectious influenza units ext,racted from the GUI resembled t,hnt reported for the related incomplete fowl plague virus obtained in the same manner (Schafer et aZ., 1954). It may be suggested, therefore, t)hat incomplete virus, freed on destruction of the infected host cell or by mechanical disintegration of the latter, may represent a different st,ate of organizat,ion than that of noninfectious forms released over a prolonged period of time from the chorioallantoic membrane (Paucker and Henle, 1955). It is not known, however, whether incomplete particles obtained in t.he course of serial
SECTIOKS
OF
IKFECTIOUS
AM)
&-ONNFECTIOUS
PARTICLES
37
undiluted passages or from a partially heat-inactivated seed represent intermediates which have been blocked at a more advanced stage in the reproductive process or whether they may be aberrant forms altogether. The morphology and dimensions of sectioned infectious particles confirm earlier observations of Morgan et al. (1956)) who examined sections of infected chorioallnntoic membranes. The absence of electron-dense material in t,he interior of incomplete preparations fit,s in well with other data on the chemical nature of the virus and on the basic structural components which can be released by treatment with ether (Hoyle, 1950, 1952). Ada and Perry (1956) showed that incomplete influenza virus may carry only one-third the amount of nucleic acid extractable from infectious units. All of the latter is contained within the soluble antigen portion (Hoyle et al., 1954). Thus, Lief and Henle (1958b) demonstrated that various t.ypes of noninfectious virus incorporated lesser quantities of S antigen than standard virus. This was confirmed in the preceding and present papers. However, inasmuch as the nucleoprotein has been calculated to constitute only about 14 % of t’he weight of the intact infective particle, it is felt t.hat. a reduced incorporation of this component, to the extent noted, could not alone account for the marked differences in electron density observed. As mentioned in the first paper, the basic HA unit of influenza virus consists of spherical particles measuring from 35 to 40 rnp in diameter. While many of these can be released from infectious particles, incomplete virus appears to be largely deficient in this component. Units of this size would be expected to occupy a considerably greater portion of the interior and mass of the elementary body than S antigen. The lack of electron-dense matter noted in sections is, therefore, likely to be due to a reduced incorporation of the HA constituent into t,he incomplete virus structure. Ultrathin sect,ions prepared from suspensions of HeLa and membrane viruses were often difficult to interpret. In both, amorphous structures predominated. However, this finding W~LSnot unexpected since spray drop preparat’ions had revealed that these materials contained mainly virus ghosts, apparently devoid of any structural rigidity. Repeated attempts in this laboratory at sectioning ghostlike structures and hemagglutinating components obtained on treatment. of sttmdard virus elementary bodies with ether resulted in precisely the sarne picture. The outer structures of incomplete units, however, do not; appear to differ much from those of the infectious agent. Because virus filaments
38
BIRCH-ANDERSEN
AND
PAUCKER
have also been shown to lack any internal structure throughout most of their length, the possibility cannot be excluded that some of the hollow spheresmay represent, cross sections of filaments. However, the number of the latter is small in relation to the total number of particles (lessthan 5%) and their distribution is the same in infectious and in incomplete materials. Thus the majority of cross sections would represent, spherical particles rather than filaments. Attempts at solving this question by serial sections have failed becausethe lack of reference sites made it impossible to identify a given field in consecutive sections. A component lacking an electron-dense interior and thus of similar appearance as incomplete virus was occasionally encountered in fully infectious materials. It was mainly present in the upper layers of the virus pellet used for sectioning. Thus it is possible that even standard virus obtained under optimal conditions of growth (Horsfall, 1954; E’inter et al., 1955) may cont,ain a certain number of incomplete urms, as suggestedby von Magnus (1952). This observation would also be compatible with studies in the analytical centrifuge which indicated that standard virus often contained small amounts of a component sedimenting at a rate similar to that of incomplete virus (Gard and van Magnus, 1946; Gard et al., 1947; Gard et al., 1952). Heat-inactivated standard virus reveals some morphological changes presumed to be due to protein denaturntion during prolonged exposure in vitro to 37”. These appear to involve mainly the surface of the virus. The dense imler structure of these particles is maintained, and they resemble standard rather than incomplete virus. This is also in accordance with its behavior in the analytical centrifuge (Gard et al., 1952). In addition, the fact that it contains similar amounts of soluble antigen as the infectious agent indicates also that the inactivat’ed virus represents an entity distinct from the other noninfectious forms. ACKNOWLEDGMENTS The
authors gratefully acknowledge Mrs. Helene Ravn, 1Mrs. Kirsten Diemer, also due to Miss Anne-Grete Ovcrgaard material.
the competent technical and Mrs. Henny Jacobsen. for expert handling of the
assistance of Thanks are photographic
REFERENCES AUA, G. L., and PERRY, B. T. (1956). between biological characteristics nucleic acid. J. Gen. Microbial. 14, BACKUS, R. C., and WILLIAMS, It. C. volatile suspending media in the croscopy. J. Appl. Phy8. 21, 11-15.
Influenza virus nucleic acid: Relationship of the virus particle and properties of the 623633. (1950). The use of spraying methods and of preparation of specimens for electron mi-
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NONINFECTIOUS
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I~IRCH-ANDERSEN, A. (1956). Unpublished results. I~AWSON, I. hr., and ELFORD, W. J. (1949). The investigation of influenza, and related viruses in the electron microscope by a new technique. J. Gen. Microbial. 3, 298-311. EPSTEIN, M. A. (1958). Observations on the structure and composition of mature vaccinia virus and the significance, with regard to its multiplication, of free nucleic acid isolated with it. Abstracts ‘Yth Intern. Cov~gr. Microbial. Stockholm 1968, pp. 233-234. FINTER, N. B., Lru, Cl. 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, 461-478. GARD, S., and VON MAGNUS, P. (1946). Studies on interference in experimental influenza. II. Purification and centrifugation experiments. Arkiv Kemi, Mineral. Geol. 24, 1-t. GARD, S., VON MAGNCS, I’., and SVEDMYR, A. (1947). Physico-chemical aspects on inhibition and interference in experimental influenza. 4th Intern. Congr. Microbiol. Copenhagen, 1947 Section III, 301-302. GARD, S., VON MAGNUS, P., SVEDMYR, A., and BIRCH-ANDERSEN, A. (1!)52). St’udies on the sedimentation of influenza virus. Arch. Virusforsch. 4, 591611. GRANOFF, A. (1955). Soninfectious forms of Newcastle disease and influenza viruses. Studies of noninfectious virus occurring within cells that are producing fully infectious virus. Virology 1, 516532. HENLE. G., GIRARDI, A., and HENLE, W. (1955). A non-transmissible cytopathogenic effect of influenza virus in tissue culture accompanied by formation of non-infectious hemagglutinins. J. Exptl. Med. 101, 25-41. HENLE, W. (1953). Developmental cycles in animal viruses. Cold Spring Harbor Symposia Quant . Riol. 18, 3544. HENLE, W., LIU, 0. C., PAIXKER, K., and LIEF, F. S. (1956). Studies on hostvirus interactions in the chick embryo-influenza virus system. XIV. The relation between tissue-bound and liberated virus materials under various conditions of infection. J. Exptl. Med. 103, 799-822. HOLL~S, I. (1957). Electronmicroscopic examination of complete and incomplete influenza virus particles. Acta Microbial. Acad. Sci. Hung. 4, 459-474. HORSFALL, I?. L., JR. (1954). On the reproduction of influenza virus, quantitative studies with procedures which enumerate infective and hemagglutinating virus particles. J. Exptl. Med. 100, 135-161. HORSFALL, F. L., JR. (1955). Reproduction of influenza viruses. Quantitative investigations with particle enumeration procedures on the dynamics of influenza A and B virus reproduction. J. Exptl. Med. 102, 441-473. HOYLE, L. (1950). The multiplication of influenza viruses in the fertile egg. J. Hyg. 48, 277-297. HOYLE, L. (1952). Structure of the influenza virus. The relation between biological activity and chemical structure of virus fractions. J. Hyg. 60, 229-245. 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. 63, 119-127. LIEF, F. S., and HENLE, W. (1956a). Studies on the soluble antigen of influenza
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Ah3
PAUC!KER
virus. I. The release of S antigen from elementary bodies by treat’ment 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, 752-797. MORGAN, C., ROSE, H. M., and MOORE, I). H. (1956). Structure and development of viruses observed in the electron microscope. III. Influenza virus. b. Exptl. iwed. 104, 171-182. PAUCKER, K., and HENLE, W. (1955). Studies on host-virus interactions in the chick embryo-influenza virus system. XI. The effect of partial inactivation of standard seed virus at 37°C upon the progeny. J. Exptl. Med. 101, 479-492. PACCKER, K., and HENLE, W. (1958). Interference between inact’ivated and active influenza viruses in the chick embryo. II. Interference by incomplete forms of influenza virus. +‘irology 6,198-214. PACCKER, K., BIRCH-ANDERSEN, A., and VON MAGNUS: P.‘(1959). Studies on the structure of influenza virus. I. Components of infectious and incomplete particles. Virology 8, I-20. PETERS, 11. (1956). Morphology of resting vaccinia virus. IVafure 178, 1453-1455. PYE, J., HOLDEN, H. F., and DONALD, H. B. (1956). Sedimentation behavior and electron microscopic examination of purified influenza virus. J. Gen. Mic,rohiol. 14, 634-636. SCHAFER, W., ZILLIG, W., and MGNK, K. (1954). Isolierung und Charakterisierung hgmagglutinierender, nicht-infektiiiser Einheiten bei klassischer Gefliigelpest. Ein Beitrag zur Kenntnis der “inkompletten Virusformen.” Z. Nafurforsch. 9b, 329-340. SHIOTA, 0. (1956). Studies on the incomplete form of influenza virus. II. The morphological character of so-called incomplete particles of virus. I’irus (Osaka) 6, 193-206. SJBsTRANn, F. S. (1953). A new microtome for ultrathin sectioning for high resolution electron microscopy. Experientia 9, 114. SJ~STRAND, F. S. (1956). Electron microscopy of cells and tissues. In Physical Techniques in Hiological Research (G. Oster and A. W. Pollister, eds.), Vol. III, pp. 241 ff. Academic Press, New York. VON MAGNUS, P. (1951a). Propagation of the PR8 strain of influenza A virus in chick embryos. I. The influence of various experimental conditions on virus multiplication. Acta Pathol. Microbial. &and. 28, 251-277. VON MAGNUS, I’. (195lb). Propagation of the PR8 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. &and. 28, 278-293. VON MAGNUS, P. (1952). Propagation of the PR8 strain of influenza A virus in chick embryos. IV. Studies on the factors involved in the formation of incomplete virus uponserial passageof undiluted virus. Actu Pathol. Microbial. &and. 30, 311-335. Voss, H., and WENGEL, E. (1955). Formen des Grippevirus. Zenk. Bakteriol., Parasitenk. Abt. Z Orig. 162, 225-235. WERNER, G. H., and SCHLESINGER, R. W. (1954). Morphological and quantitative comparison between infectious and non-infectious forms of influenza virus. J. Exptl. Med. 100, 203-216.
8,214O
Studies II. Ultrathin
(1959)
on the Sections
Structure
of Infectious
AKSEL BIRCH-ANDERSEN The Laboratory
of Biophysics
of Influenza and Noninfectious
Particles’
AND KURTPAUCKER~'~
and InfEuenza Department, Copenhagen, Denmark Accepted
Virus
December
State
Serum
Institute,
8, 1958
The morphology of infectious and heat-inactivated influenza virus, as well as of incomplete particles obtained under various conditions, was studied in the electron microscope. Purified preparations of elementary bodies were examined in ultrathin sections and in sprays from a volatile suspending medium. The composition of the materials under study was determined by relating infectious titers to hemagglutinin content and by assaying for the amount of internally bound soluble antigen released on treatment with ether. Standard virus in spray drop preparations consisted of spherical particles of uniform size and measuring on the average 118 rnp in diameter. Sections revealed spheres with a mean diameter of 70 rnp which contained an electrondense core limited by an inner membrane, an external coat of lower density, and a less distinct external membrane. Sprays of incomplete virus derived from serial undiluted passages (UP) and from the inoculation of heated standard seed (ASTP), contained particles of variable sizes and flatter than infectious virus, as well as large baglike structures. In addition to these, completely flattened and apparently empty ghosts were encountered with virus grown in HeLa cells and with noninfectious elementary bodies extracted from chorioallantoic membranes of standard eggs (CAM). In sectioned particles of UP, ASTP, and HeLa-grown virus, electron-dense centers were usually lacking and no internal details could be discerned. Their outer structures did not differ essentially from those of the infectious agent. Sections of preparations extracted from chorioallantoic membranes containing considerable quantities of noninfectious hemagglutinins revealed mainly masses of amorphous material. Those viral structures which could be recognized, however, resembled the other incomplete viruses described. 1 A portion of this report was presented Microbiology in Stockholm, 1958. 2 Fellow of the National Foundation for 3 Present address: Research Department, delphia, Philadelphia, Pennsylvania. 21
at the 7th Infantile The
International
Paralysis. Children’s
Hospital
Congress
of Phila-
of
22
BIRCH-ANDERSEN
Heat-inactivated standard virus, longed exposure to 37”C, possessed of the infectious agent. It is thought, from other noninfectious forms.
AND
PAUCKER
although somewhat changed due to proall the internal structural characteristics therefore to represent an entity distinct
INTRODUCTION
In the past, noninfectious forms of influenza virus obtained from mouse brain, from serial undiluted passage in eggs, from egg passage of mixtures of heat-inactivated and infectious virus and from chorioallantoic membranes have been examined in the electron microscope (Werner and Schlesinger, 1954; Voss and Wengel, 1955; Pye et al., 1956; Shiota, 1956; Hellos, 1957). In most of these studies, particles adsorbed onto lysed red cell ghosts according to a technique originally described by Dawson and Elford (1949) were observed. They have been described as flattened pleomorphic structures, somewhat larger in diameter than the infectious agent and of lesser density. The preceding study (Paucker et al., 1959) revealed that on treatment of incomplete virus units with ether (Hoyle, 1950, 1952), only small numbers of structural components identified with soluble (S) antigen activity were released. This corroborated the serological findings of Lief and Henle (1956b) and the determinations of nucleic acid content by Ada and Perry (1956). In addition, the data suggestedthat fewer than normal spherical hemagglutinating components may have been incorporated into the incomplete virus struct,ure. The dimensions of the HA subunits made it seem likely that they, rather than S antigen, were responsible for the altered appearance of the noninfectious agent. This pointed to fundamental differences in the internal organization of the two forms of virus, and these have been studied further, making use of recent advances in preparatory techniques for electron microscopy. The examination of whole particles appeared of limited value in such an investigation. On the other hand, thin sections have been used to advantage in the study of vaccinin virus (Peters, 1956; Epstein, 1958) and of bacteriophage (Birch-Andersen, 1956). This method was adapted to permit the examination of ultrathin sect,ions of purified virus materials obtained under the conditions favoring the production of maximal amounts of infectious virus (Horsfall, 1954; Finter et al., 1955) or those giving rise to incomplete forms. As a source of the latter the embryonat’ed egg, which is potentially capable of producing infectious as well as noninfectious virus, and the HeLa cell known to support an incomplete cycle of growth only (Henle et al., 1955) were
SECTIOKS
OF
ISFECTIOUS
AND
NONINFECTIOUS
PARTICLES
23
selected. Since one cannot assume a priori that the same fundamental processes underlie the production of incomplete virus by various laboratory manipulations and from different sources, morphological dissimilarities among incomplete virus populations arising from different host systems might not be unexpected. The inclusion of standard virus rendered largely noninfective by heating in vitro at 37” was motivated by the hypothesis that incomplete forms may represent originally infect,ious units which were thermally inactivated in 02’0 during the incubation period (Horsfall, 1954, 1955). MATERIALS
.4ND METHODS
Virus. The PR8 strain of influenza A was used in this study. Standard virus (ST) was obtained by inoculating allantoically 11- to 13-day-old embryonated eggs with lo3 to lo4 ID,, of seed virus. The eggs were chilled and harvested after 24 hours of incubation at 37”. Undiluted passage (UP) seed was prepared essentially by the method of von Magnus (195lb). Heat-inactivated virus @ST) was obtained by incubating standard seed in rubber-stoppered centrifuge bottles in a 37” water bath for several days. Inoculation of the undiluted partially heatinactivated preparation yielded heat,ed passage virus (ASTP) as described earlier (Paucker and Henle, 1955). Viruses derived from HeLa cells were prepared as reported previously (Paucker and Henle, 1958). In order to obtain sufficiently pure preparat,ions of HeLa virus, only hemagglutinins released into the fluid medium from destroyed cells, but not those derived from mechanical disintegration of cell debris, were used for further handling. Membrane virus (CAM) was derived from chorioallantoic membranes of eggs inoculated with 103to IO4 EID,, of standard seed. The membranes were harvested after 24 hours of incubatlion at 37” and washed repeatedly in large volumes of 41/100 phosphate-buffered saline solution (BSS) at pH 7.4 until they were free of blood. They were suspended in BSS cont’aining 5% RDE,” using 1 ml per membrane, and incubat,ed overnight in a 37” water bath. After further washing, the membranes, resuspendedin the same volume, were emulsified in chilled Servall omnimixer vesselsimmersed in ice. Cellular debris was removed by low-speed centrifugation and hemagglutinins were purified and concentrated by repeated adsorptions onto and elutions from chicken red cells. 4Receptor-destroyingenzyme; obtained from the Behring Werke, Max-burg, Germany.
24
BIRCH-ANDERSEN
AND
PAUCKER
Adsorption and elution of virus. All preparations were subjected to two or more cycles of adsorption and elution during which twenty- to fiftyfold concentration of virus was attempted. Thrice washed and packed chicken cells were added to the chilled materials to constitute a final concentration of 1 to 5%. The mixtures were incubated in the cold for 1 hour and the red cells were removed by centrifugation. After being washed once in ice-cold saline solution, they were resuspended in appropriate volumes of phosphate-buffered saline containing per milliliter approximately 50 units of RDE, 200 units of penicillin, and 200 pg of streptomycin. Elution of virus was carried out for 2 hours in a 37” water bath. The red cells were then removed by low-speed centrifugation and the supernatant fluid contained the elementary body suspension. Centrifugation. All elementary body suspensions, after having undergone preliminary purification by at least two cycles of adsorption and elution, were subjected to two or more sedimentations in the Spinco Preparat,ory L centrifuge. Standard, UP, AST, and ASTP preparations were spun for 60 minutes at 51,000 g, and HeLa-grown as well as CAM viruses were centrifuged for 180 minutes at 105,000 g. After each centrifugal run, sediments were resuspended in appropriate volumes of phosphate-buffered saline and clarified for 10 minutes at 6600 g. Assays for virus activity. Egg infectivity and hemagglutination tests were performed as described earlier (von Magnus, 1951a). The amounts of internally bound soluble antigen were determined by subjecting elementary body suspensions to treatment wit’h ether as described in the preceding report. The techniques used for complement fixation tests (Henle et al., 1956) as well as the preparation of anti-V and anti-S guinea pig sera (Lief and Henle, 1956a) have been reported. Preparation of sections. The concentrations of individual virus materials were adjusted to approximately 104.5 hemagglutinin units per milliliter. They were sedimented once more at the same speeds as those used during earlier cenOrifuga1 runs. After evacuating the supernatant fluid by gentle suction, the virus pellets were removed with a scalpel and embedded in 1.8 % Difco agar made up in phosphate-buffered saline of pH 7.4. For this purpose plastic cylinders, 2 cm in diameter and 1.5 cm in height, had been constructed through which two holes measuring 0.5 cm in diameter had been drilled. These were blocked at one end with rubber stoppers and filled to the half-way mark with agar which was permitt,ed to solidify. The virus pellets were deposited on top of the latter and the molds were then completely filled with agar cooled down
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
25
to approximately 50”. After hardening in the cold, the stoppers were removed and the agar cylinders were pushed out by means of a metal rod. Only the center section containing the virus pellet was used for further processing. Essentially, the method described by Sjijstrand (1956) was followed, except that fixation was carried out for 1 hour at room temperature in 1% osmium tetraoxide made up in phosphate-buffered saline at pH 7.4. Most of the agar surrounding the pellets was carefully peeled away before embedding small portions of the latter in prepolymerized methacrylate. A Sjiistrand ultramicrotome (Sjbstrand, 1953) was used for cutting the sections, which were floated on a 20% ethyl alcohol-80 % water mixture before being deposited on Formvar-coated grids. The sections with methacrylate left in place were examined in a Philips electron microscope, model EM 100 B. The stigmator of the compensated pole piece of the instrument was routinely adjusted so that the degree of astigmatism was of the order of 0.3-0.4 mp. Spraying of virus. This technique has been described in the preceding paper (Paucker et al., 1959). EXPERIMENTAL
Selection of Virus Preparations The suitability of the materials for study in the electron microscope was determined by infectivity and hemagglutination tests. The degree of incompleteness was expressed as IDr,o:HA ratios. Complement fixation titrations with anti-V and anti-S guinea pig sera (Lief and Henle, 1956a) were performed on samples of elementary body suspensions after concentration and purification by adsorption onto and elution from chicken red cells, but prior to sedimentation in the centrifuge. As a rule none of the purified preparations reacted with the anti-S serum prior to ether treatment (EB) whereas varying amounts of soluble antigen were released after this procedure (EEB). These values were related to the HA titers of the original concentrates and expressed in terms of hemagglutinating units required to yield 1 complement-fixing S unit on treatment with ether (HA:S ratio). Table 1 lists the pertinent data used to evaluate the composition of the materials selected. Fully infectious virus grown under standard conditions (ST) exhibited ID,,: HA and HA:S ratios typical for this material. In the order of 106.271D50were equivalent to 1 HA unit, and 75 of the latter released 1 complement-fixing S unit on exposure to the solvent. The IDS,: HA ratios of a fourth undiluted passage (UP4), a single undiluted passage of stand-
26
BIRCH-ANDERSEN
AND
TABLE THE
COWOSITION
Type of PR8 Titus
OF VIRUS
ST
Infectious
1
PREPARATIONS 1Dso:HA 10s
Composition
!
6.27
i
PAUCKER
SELECTED
FOR Titers
hlaterial tested
CFV
602,500 870,000
STUDY
per milliliter
HA
EBd EEB*
MICROSCOPIC
6,400 8,000
i
CFS
EBHA:S --
75 ---
UP4
Incomplete
4ASTP
2.70
Incomplete
2.99
.HeLa
Incomplete
i
3.65”
-I-
CAM
Incomplete
4.17
EB EEB .,----EB EEB -~-~EB EEB ----,---.--i-_-‘- EB EEB
43,100 43,100 ---~-
640 480 ---__
15,150 7,590
160 80
160 60
320 320
i
----
---11,210 7,590
10 60
---_ j ?J.D.c
1 K.D.
720
---_ 505
35” -.--
1 x.1).
4AST 94 n So increase over ID,, titer of residual b No increase over external S titer before c Kot, determined. d Elementary body. e Ether-treated elementary body.
seed. ether
treatment.
ard virus inactivated for 4 days at 37” (4ASTP) and of virus derived from chorioallantoic membranes of eggs infected with diluted seed (CAM) were all subst,antially lower. This indicated that predominantly noninfectious particles were present. In agreement with earlier data (Lief and Henle, 195610) approximately ten times more HA units of UP4 and seven times more HA units of 4ASTP than of ST were needed to free 1 soluble antigen unit. In the case of HeLa virus, no increase whatever in infectious titer over that of the residual seed inoculum was noted. Repeated cycles of purification with chicken red cells could not remove all of the external soluble antigen, but no additional amounts were released by ether. The ID50:HA ratio of 4AST indicates that most of its initial infectivity had been destroyed in the course of heating. The content of S antigen, however, was of a similar order as that found in standard virus.
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
27
Virus Sprays All virus materials were sprayed prior to sectioning in order to assess their purity and to check on the morphology of whole particles. ST virus (Fig. 1) consists of spherelike units with a mean diameter of 118 rnp. While some variation in shape can be noted, their diameters fall within a narrow range. In many instances a rimlike structure, approximately 15 rnp in thickness, can be seen surrounding the virus particles. Incomplete virus, on the other hand, by whatever method produced, represents a more heterogeneous aggregation of particles, exhibiting considerable variations in shape and size. In both UP4 virus (Fig. 2) and the lASTP preparation (Pig. 3) the particles appear somewhat flattened. Ghostlike structures with well-defined rims can also be seen. The latter predominate in both the HeLa (Fig. 4) and CAM (Fig. 5) preparations. In addition, the former contains also particles approximately 9@-100 rnp in diameter which are characteristically arranged in short chains. The heat-inactivated virus (4AST) is shown in Fig. 6, a and b. The outline of these particles is not as well defined as standard virus, and they show a tendency toward clumping. These changes are probably due to mild denaturation during the process of heating. Virus Sections The morphological differences between infectious and incomplete influenza virus are more clearly brought out in ultrathin sections. Standard virus units (Fig. 7) appear in general as spherical or ellipsoidal bodies, often somewhat compressed during the cutting process. When the plane of sectioning cuts cent.rally through a particle, a dense inner region is revealed, measuring on the average 45 rnp in diameter, which is limited by a membrane approximately 40 B in thickness. This structure is surrounded by a zone of lower density about 10 mp in thickness, which appears to be enclosed by a less clearly defined outer membrane. Averaging the long and short axes of the compressed particles, introducing thereby a negligible error (in the order of 1% in this case), t’he sectioned units of standard virus have a diameter of about 70 mp. A certain number of filaments is also present in this preparation. Their outer limiting structures resemble that of the spherical particles and their diameter is of t’he same order or slightly less in most, instances. In the interior no structural detail can be discerned except at one end where electron-dense mat,erial is condensed in an area approximat,ely the size of a free virus particle. The higher magnification (Fig. 8) shows
FIGS.
l-4
1. Standard virus. Spherical elementary bodies. 2. Incomplete virus after four serial undiluted egg passages. Particles of size and shape. 3. Incomplete virus after egg passage of heated standard seed. Note appearance of particles and some filamentous forms. 4. Incomplete virus from HeLa cells. Ghosts and small flattened spherical sometimes arranged in chains. FIG.
FIG.
variable FIG.
flattened FIG.
particles, FIGS.
shadowed.
l-4. Sprayed from ammonium Magnification: X 30,000.
acetate 28
buffer.
Palladium-platinum
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
29
FIGS. 54 a-b FIG. 5. Incomplete virus from chorioallantoic membranes producing infectious virus. Ghosts and clumped flattened spheres. FIG. 6. a and b. Heat-inactivated standard virus. Note blurred outlines of particles and tendency toward clumping. FIGS. 5 and 6. Sprayed from ammonium shadowed. Magnification: X 30,000.
acetate buffer. Palladium-platinum
that the material in the interior of the spherical standard particles is not uniformly arranged. It has a patchy appearance and it is divided into small compartments by strands of higher electron density. Sections of the UP4 preparation are shown in Fig. 9. These particles show a considerable distribution in sizes, with diameters ranging from
SECTIONS
OF
INFECTIOUS
FIG. 8. Sections of sbandard ance of centers. Magnification:
AND
virus. Note X 225,000.
NONINFECTIOUS
uneven
density
31
PARTICLES
and patched
appear-
32
BIRCH-ANDERSEN
FIGS. FIG.
varying FIG.
standard
AND
PAUCKER
9-10 a-b
9. Sections of incomplete fourth undiluted passage virus. Particles of sizes lacking electron-dense centers. Magnification: X 94,500. 10. a and b. Sections of incomplete virus obtained on passage of heated virus. Note absence of dense centers. Magnification: X 94,500.
FIGS. 11 a-c-12 FIG. 11. a-c. Sections of incomplete virus grown in HeLa cells. Aggregates of particles lacking dense centers. Magnification: X 150,000. FIG. 12. Sections of incomplete virus from chorioallantoic membranes. Amorphous material and some hollow virus structures. Magnification: X 94,500. 33
34
BIRCH-ANDERSEN
AND
PAVCKER
50 to 120 rnp in accordance with the micrographs of sprayed intact units. The particles are in general more compressed and most of them reveal little or no detectable internal structure; the over-all impression is that of hollow virus units. However, some spheres with electron-dense centers are also present. They may conceivably represent infect’ious particles inasmuch as incomplete preparations always possess a certain degree of infectivity. Whenever the plane of section is optimally located, two outer rings are discernible, which suggests that the limiting structures of this virus resemble that of the infectious agent. The balloonlike forms, on the other hand, are less clearly defined and may be in a stage of disintegration. Essentially a similar appearance is presented by 4ASTP
FIG.
centers
13. Sections and blurred
of heat-inactivated outlines. Magnification:
standard virus. X 94,500.
Note
electron-dense
SECTIONS
OF
INFECTIOUS
AND
NONINFECTIOUS
PARTICLES
35
(Fig. 10, a and b), except that the integrity of the virus structure was not as well preserved. Many aberrant forms, in a more or less disrupted state can be seen. Most of the smaller units with well-defined limiting membranes exhibit centers of low electron density. The outlines of HeLagrown virus (Fig. 11, a, b, and c) are somewhat blurred. However, purification of this mat,erial met with considerable difficuhy, as can be inferred from the micrograph of the sprayed preparation and the presence of external soluble antigen which could not be removed during repeated red cell adsorption and elution cycles. The particle diameters range mostly from 60 to 65 rnF and are thus slightly smaller t’han those of standard virus. The units show a tendency to form aggregates. Most, of them, like the other incomplete preparations, show no internal structure. Sectioning of the CAM preparation (Fig. 12) did not yield readily interpretable results. Most of the material seems amorphous and may consist in part of host material carried through the purification procedure. However, wherever units of virus morphology are recognizable, their centers are not filled with electron-dense material and thus they resemble incomplete rather than standard virus. Heat-inactivated virus (Fig. 13), on the other hand, differs considerably from the incomplete preparations studied. The particles, are fairly uniform in size, and their mean diameter is of a similar order as that of ST virus (71 mp). They also exhibit centers filled with a material of high density and the same internal patched appearance as that encountered in the infectious forms. The limiting coats cannot be recognized as clearly, but the hazy outlines may well be due to denaturation during prolonged exposure to 37”. DISCUSSION
The present study indicated that the internal structure of incomplete influenza virus particles, obtained by various manipulations and from two distinct hosts, differed markedly from that of the infectious form. Ultrathin sections of purified suspensionsof infective units revealed t,hat their centers were filled with an electron-dense material which showed a certain amount of organization. Sectioned incomplete particles obtained from serial undiluted passages(von Magnus, 1951b), the inoculation of heated standard seed (Paucker and Henle, 1955) and from HeLa cells (Henle et al., 1955) differed in one important aspect from standard virus. Electron-dense centers mere usually absent and t,hus no internal structural details could be discerned. Essentially the same held true for non-
36
BIRCH-ANDERSEN
AR;D
PAUCKER
infectious units derived from chorioallantoic membranes producillg infective virus (Henle, 1953; Granoff, 1955; Henle et al., 1956). However, only few virus structures could be identified with reasonable cerbainty within the amorphous mat’erial seen in sections. The examinat’ion of intact’ particles was carried out simultaneously. Whereas infectious elementary bodies a.ppeared as well-defined spherical units, incomplete forms derived from undiluted serial passages or from a partially preinactivated inoculum exhibited a greater assortment of sizes and sha.pes and a certain degree of flattening. This confirms in general the reports of others (Voss and Wengel, 1955; Shiota, 1956; Pye et al., 1956; Hellos, 1957). The differences in morphology observed were perhaps not as striking as those cited earlier, but the spraying from volatile suspending medium (Backus and Williams, 1950) may have minimized to some extent the distortions which occur during the conventional air-drying of preparations for microscopic st,udy. Whole incomplet’e particles obtained from HeLa cells or extracted from chorioallantoic membranes producing standard virus differed markedly from both the infectious and the other incomplete forms mentioned. They consisted largely of ghostlike structures and aggregates or chains of small, flat,tened units. These findings are at variance with observations from ot,her laboratories. Werner and Schlesinger (1953) found that part,icles obtained from rnembranes and allantoic fluids of eggs in which standard virus was produced were alike in appearance. Shiota (1956) described a narrower distribution of sizes, slightly smaller diameters, but an otherwise normal morphology of the virus derived from the CAM. However, the IDbo:HA rat)ios of their prepambions (lOfi,O or more) and the method of ext,ruction indicate t)hat’ not sufficient care may have been t’aken in removing infectious virus which, after migration through t’he host cell membrane, had remained superficially attached t’o it. On the other hand, the morphology of noninfectious influenza units ext,racted from the GUI resembled t,hnt reported for the related incomplete fowl plague virus obtained in the same manner (Schafer et aZ., 1954). It may be suggested, therefore, t)hat incomplete virus, freed on destruction of the infected host cell or by mechanical disintegration of the latter, may represent a different st,ate of organizat,ion than that of noninfectious forms released over a prolonged period of time from the chorioallantoic membrane (Paucker and Henle, 1955). It is not known, however, whether incomplete particles obtained in t.he course of serial
SECTIOKS
OF
IKFECTIOUS
AM)
&-ONNFECTIOUS
PARTICLES
37
undiluted passages or from a partially heat-inactivated seed represent intermediates which have been blocked at a more advanced stage in the reproductive process or whether they may be aberrant forms altogether. The morphology and dimensions of sectioned infectious particles confirm earlier observations of Morgan et al. (1956)) who examined sections of infected chorioallnntoic membranes. The absence of electron-dense material in t,he interior of incomplete preparations fit,s in well with other data on the chemical nature of the virus and on the basic structural components which can be released by treatment with ether (Hoyle, 1950, 1952). Ada and Perry (1956) showed that incomplete influenza virus may carry only one-third the amount of nucleic acid extractable from infectious units. All of the latter is contained within the soluble antigen portion (Hoyle et al., 1954). Thus, Lief and Henle (1958b) demonstrated that various t.ypes of noninfectious virus incorporated lesser quantities of S antigen than standard virus. This was confirmed in the preceding and present papers. However, inasmuch as the nucleoprotein has been calculated to constitute only about 14 % of t’he weight of the intact infective particle, it is felt t.hat. a reduced incorporation of this component, to the extent noted, could not alone account for the marked differences in electron density observed. As mentioned in the first paper, the basic HA unit of influenza virus consists of spherical particles measuring from 35 to 40 rnp in diameter. While many of these can be released from infectious particles, incomplete virus appears to be largely deficient in this component. Units of this size would be expected to occupy a considerably greater portion of the interior and mass of the elementary body than S antigen. The lack of electron-dense matter noted in sections is, therefore, likely to be due to a reduced incorporation of the HA constituent into t,he incomplete virus structure. Ultrathin sect,ions prepared from suspensions of HeLa and membrane viruses were often difficult to interpret. In both, amorphous structures predominated. However, this finding W~LSnot unexpected since spray drop preparat’ions had revealed that these materials contained mainly virus ghosts, apparently devoid of any structural rigidity. Repeated attempts in this laboratory at sectioning ghostlike structures and hemagglutinating components obtained on treatment. of sttmdard virus elementary bodies with ether resulted in precisely the sarne picture. The outer structures of incomplete units, however, do not; appear to differ much from those of the infectious agent. Because virus filaments
38
BIRCH-ANDERSEN
AND
PAUCKER
have also been shown to lack any internal structure throughout most of their length, the possibility cannot be excluded that some of the hollow spheresmay represent, cross sections of filaments. However, the number of the latter is small in relation to the total number of particles (lessthan 5%) and their distribution is the same in infectious and in incomplete materials. Thus the majority of cross sections would represent, spherical particles rather than filaments. Attempts at solving this question by serial sections have failed becausethe lack of reference sites made it impossible to identify a given field in consecutive sections. A component lacking an electron-dense interior and thus of similar appearance as incomplete virus was occasionally encountered in fully infectious materials. It was mainly present in the upper layers of the virus pellet used for sectioning. Thus it is possible that even standard virus obtained under optimal conditions of growth (Horsfall, 1954; E’inter et al., 1955) may cont,ain a certain number of incomplete urms, as suggestedby von Magnus (1952). This observation would also be compatible with studies in the analytical centrifuge which indicated that standard virus often contained small amounts of a component sedimenting at a rate similar to that of incomplete virus (Gard and van Magnus, 1946; Gard et al., 1947; Gard et al., 1952). Heat-inactivated standard virus reveals some morphological changes presumed to be due to protein denaturntion during prolonged exposure in vitro to 37”. These appear to involve mainly the surface of the virus. The dense imler structure of these particles is maintained, and they resemble standard rather than incomplete virus. This is also in accordance with its behavior in the analytical centrifuge (Gard et al., 1952). In addition, the fact that it contains similar amounts of soluble antigen as the infectious agent indicates also that the inactivat’ed virus represents an entity distinct from the other noninfectious forms. ACKNOWLEDGMENTS The
authors gratefully acknowledge Mrs. Helene Ravn, 1Mrs. Kirsten Diemer, also due to Miss Anne-Grete Ovcrgaard material.
the competent technical and Mrs. Henny Jacobsen. for expert handling of the
assistance of Thanks are photographic
REFERENCES AUA, G. L., and PERRY, B. T. (1956). between biological characteristics nucleic acid. J. Gen. Microbial. 14, BACKUS, R. C., and WILLIAMS, It. C. volatile suspending media in the croscopy. J. Appl. Phy8. 21, 11-15.
Influenza virus nucleic acid: Relationship of the virus particle and properties of the 623633. (1950). The use of spraying methods and of preparation of specimens for electron mi-
SECTIONS
OF
IKFECTIOUS
AND
NONINFECTIOUS
PARTICLES
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