J. gen. Virol. 0975), 27, 313-3x8
313
Printed in Great Britain
A Morphological Study of the Internal Component of
Influenza Virus
By J U N E D. A L M E I D A
AND C. M. B R A N D
Department of Virology, Wellcome Research Laboratories, Langley Court,
Beckenham, Kent, BR3 3BS
(Accepted 28 January I975)
SUMMARY
Rapid treatment of influenza virus directly on the microscope grid with non-ionic
detergent has allowed better visualization of the internal component. Many
micrographs show that this ribonucleoprotein (RNP) is present as a continuous
strand of 6 nm diam. arranged in the form of a double coil or helix. In spite of the
minimal treatment to which the virus was subjected most helices still showed signs
of degradation. The findings that we have obtained lead us to suggest that the RNP
component of influenza virus must be very sensitive to both chemical and physical
manipulations, any of which could cause it to fracture from one continuous
strand into several pieces, although such breakages could possibly occur at specific
points along its length.
INTRODUCTION
In I96O Horne et al. described the external appearance of influenza virus and the distinctive projection-covered spherical 'and filamentous particles are now well known.
However, unlike the paramyxovirus group, there has not yet been basic agreement as to the
morphology of the internal component of influenza virus. This is, in part, due to the nature
of the influenza virus envelope, as few spontaneously disrupted particles occur in a normal
preparation of influenza virus but also in part it is due to the fact that biochemical findings
have postulated a fragmented genome (Duesberg, 1968; Pons & Hirst, I968; Skehel, I97r).
The present study describes a simple and rapid degradation technique that can be carried out
on influenza virus particles directly on the microscope grid (Nermut, I97o). The findings
obtained suggest that the major problem in establishing the morphology of the internal
component of influenza virus lies in its extreme fragility. Using this technique influenza
virus can be shown to contain an internal strand of 6 nm diam. which frequently exists
in the form of a double coil or helix.
METHODS
Viruses. Influenza virus of strains Ao/PR8/34, Ao/Bel/42, fowl plague virus, X3I and
MRC2 (a recombinant between A/Eng 42/72 and Ao/PR8/34), were grown in embryonated
eggs and purified by density gradient sedimentation, essentially as described by Skehel &
Schild (I970. The virus suspension was adjusted to a final protein concentration of Io mg/
ml. in phosphate buffered saline.
Electron microscopy. A drop of purified virus was placed on a 4oo mesh carbon-formvar
coated grid and excess fluid withdrawn with filter paper so that the grid surface was covered
by a thin layer. Before this layer could dry a drop of I ~ Nonidet (Shell Chemicals U.K.
21-2
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J . D . A L M E I D A A N D C. M. B R A N D
Fig. I. Shows the appearance of the purified influenza virus (Ao/PR8) stained directly on the
microscope grid. Note the arrowed particle in this micrograph; within it can be seen a helical
structure.
Fig. 2. The same preparation as Fig. I but after treatment on the grid with Nonidet. The particles are
now collapsed and the internal helical component can be seen. The particle at the top left of the
micrograph shows this component emerging in the form of two parallel strands.
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Internal component of influenza virus
3 T5
Ltd) was added and immediately withdrawn as before, by touching the edge of a piece of
filter paper to the grid. As quickly as possible the grid was then washed with several changes
of 1.5 ~ phosphotungstic acid adjusted to pH 6. After removing excess stain the grid was
allowed to dry and examined in a Philips 3oo electron microscope at a plate magnification
of 57ooo times. Control grids were prepared by omitting the detergent step and simply
treating the virus with the 1.5 ~ phosphotungstic acid.
RESULTS
Control grids showed large numbers of typical influenza virus particles and, although a
few elongated particles could be found, the vast majority of the virus was present as approximately spherical but pleomorphic particles in the size range 6o to 80 nm in diam.
(Fig. 0- Careful examination revealed a small number of particles that had been penetrated
by stain and in these a filamentous internal component could be seen. However, such particles were few in number and it was not possible to establish the arrangement of the internal
component beyond the fact that it appeared to be helical in nature. The detergent treated
grids on the other hand showed particles which were flattened, disrupted, and lacked the clear
cut morphology of the control virus. Examination of the background showed that surface
projections had been detached from the particles and were present either as small rosettes
or individual subunits.
The most striking feature of the detergent treated grids was the presence of numerous
coiled or helical structures in association with the majority of disrupted virus particles
(Fig. 2). These coils were most frequently seen near, but not within, disrupted envelopes,
although careful examination revealed a small number of particles where the helix was still
either completely or partially contained within the outer structure. The helices themselves
showed a wide size range both as regards diam. and overall length of the filament. However,
the majority had an overall diam. of 5° to 6o nm. Although it is difficult to give an absolute
length distribution, because of the extreme fragility of the structures, filaments of up to
2 # m have been found. Most helices were in an expanded state and it was possible to examine
the arrangement of the 6 nm strand forming the coil. The most common pattern was a
single strand in the form of a greatly expanded helix (Fig. 2). The next most frequent
arrangement was a criss-cross pattern that was subsequently interpreted as two of the strands
entwined in a double coil or helix (Fig. 3 a, b). A frequent finding was that the helical material
would exhibit the double coiled arrangement over part of the length and then, through
breakage of one strand, the single helical pattern (Fig. 4). Another pattern found was that of
two of the strands lying parallel to each other in a helical form. Fig. 5 shows this pattern
in a free helix while in Fig. z the pattern can be seen in R N P still partly contained within
the outer envelope.
In order to understand these patterns, simple models were made that could be photographed by transmission illumination. This was achieved by forming coils from strips of
celluloid which were then placed directly on the negative carrier of a photographic enlarger
and the image recorded on photographic paper. Fig. 6a and b show that a double helix
rotated about its long axis gives rise to patterns that correspond to the micrographs obtained.
The helices were extremely unstable and if there was a delay, of even a few seconds,
between applying and washing off the detergent the helices were much reduced in number.
After an exposure of 3o s they had disappeared altogether. In all the specimens examined,
various stages of degradation could be seen. Well-preserved helices were characterized by
a sharply defined filament approx. 6 nm in diam. but as degradation proceeded this dimen-
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J.D. ALMEIDA AND C.M. BRAND
Fig. 3 to 8. All virus components illustrated in this plate are derived from the X3 t strain of influenza
virus. All have the same magnification.
Fig. 3. (a) and (b) Many of the RNP helices released by detergent treatment showed the criss-cross
pattern illustrated here. The model of a double helix shown in Fig. 6 (a) exhibits the same criss-cross
arrangement.
Fig. 4. The double coils of RNP are extremely labile and here one such coil can be seen breaking down
into a single helix while the strand forming the coil can be seen expanding from a diam. of 6 nm to
one of xo nm or greater.
Fig. 5. Rotation of a double helix about its long axis reveals several different superimposition
patterns. In this instance the pattern appears as two parallel strands. This micrograph should be
compared with Fig. 6(b).
Fig. 6. (a) and (b) Transmission images of celluloid double helices showing patterns that are obtained
with two different orientations of the coil.
Fig. 7. The RNP of influenza virus is usually seen as linear fragments of IO nm diam. and up to
8o nm length. Here, fragments of this type still show the overall conformation of a helix.
Fig. 8. Frequently the helices seen remained tightly wound and it was difficult to interpret their
arrangement. However, in this micrograph it is possible to see that the coil is indeed a double helix.
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Internal component o f influenza virus
317
sion increased until diam. of greater than io nm were achieved (Fig. 5 and 7). It was also
possible to see structures that retained vestiges of helical form but which had broken down
into discontinuous segments (Fig. 7)- Conversely, a proportion of exposed coils remained
tightly wound and in a few of them it was possible to recognize the double helical arrangement (Fig. 8).
It was also found that several influenza virus strains revealed the same basic structures
but they had different levels of stability when examined by this technique. X3I and PR8
were the most stable and Bel the least stable with the Rostock strain of fowl plague and
MRC2 in an intermediate position.
DISCUSSION
The nature and structure of the internal RNP of influenza virus has given rise to considerable controversy (Duesberg, I969; Kingsbury & Webster, 1969; Pons, Schulze &
Hirst, I969; Compans, Content & Duesberg, I972; Schulze, I973). When examined by the
technique of negative staining, untreated preparations of virus contain occasional particles
that are penetrated by stain to reveal an internal component. This component appears as
a strand of approx. 6 nm diam. arranged in a helical form (Apostolov & Flewett, I965).
The helices vary considerably both as regards number of turns and overall diam. of the
coil (Almeida & Waterson, I97O). However, attempts to extract the internal component
invariably yielded preparations consisting of short lengths of a linear component of greater
than IO nm diam. and having an average length of 8o nm. Biochemical studies on both the
R N P and R N A have supported the concept that the genome of influenza virus is a fragmented one. Values ranging from 38 S to 7oS have been reported for the sedimentation
coefficient of the RNP (Duesberg, 1969; Pons et al. 1969; Compans et al. 1972). Studies on
the RNA o f influenza virus, although differing in detail, have largely agreed that when it is
extracted from the virus particle there appear to be three major size classes (Barry & Davies,
T968) containing a total of 6 or 7 pieces of R N A ( P o n s & Hirst, I968; Skehel, I970. It has
been difficult to reconcile these findings of fragmentary lengths of R N P and RNA with the
much narrower and longer structure that can be seen within spontaneously degraded particles, and which we can now demonstrate by gentle detergent disruption.
The finding of large numbers of helices must be of importance in understanding the internal
arrangement of the influenza virus but the fact that many of the helices appear to be double
may be of even greater importance in understanding this group of viruses. Comparison of
the micrographs obtained with the transmission images of the celluloid models leave little
doubt that the patterns obtained in the electron microscope were derived from double coils
or helices. Since it might be argued that strands could possibly become intertwined after
the particles had been disrupted, Fig. 2 is important as here two parallel strands can be seen
emerging from a partially disrupted particle. The extreme lability of these structures is shown
by the fact that many of the double helices have breaks in one of the two strands converting
the double coil to a single helix (Fig. 4), followed by further deterioration to the fragments
of IO nm diam. (Fig. 7).
It should be stressed that the double helical arrangement that we describe has an average
diam. of 50 to 60 nm and is formed by the coiling of the whole ribonucleoprotein strand.
This structure should not be confused with the double helix described by Compans et aL
(I972). In that instance it was suggested that a double helical arrangement could be seen
within the Io nm diam. R N P fragments and this would therefore have referred to the association of the R N A with its closely applied nucleoprotein.
The high recombination rate of influenza viruses has been well explained by the fragmented
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318
I. D. A L M E I D A
AND
C. M. B R A N D
genome concept. If, however, the present findings of a continuous R N P are substantiated it
may be necessary to re-examine this phenomenon. It may be that the continuous R N P is
a means of packaging discontinuous genetic segments and, if so, the current theory would
need little alteration. If, on the other hand, more refined techniques are able to show a continuous R N A within the RNP, other explanations must be considered to explain the high
rate of recombination. One possible explanation could be based on the double helical
configuration that we describe, as in some instances the double coil could be formed from
previously unrelated portions of RNP.
We wish to thank Mr J. Rowe for technical assistance.
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