77
,1. gen. Virol. (I97O), 9, 77-88
Printed in Great Britain
Attachment of Two Myxoviruses to Ciliated Epithelial Cells
By R. R. D O U R M A S H K I N
AND D. A. J. T Y R R E L L
Clinical Research Centre Laboratories, National Institute for Medical Research,
London, N.W. 7
(Accepted I I June I97o)
SUMMARY
It is thought that influenza and related viruses enter susceptible cells, such as
those of tissue cultures and the chorioallantois, by being adsorbed to the surface and
then taken in by an active process termed ' viropexis'. It has been suggested that this
active process resembles phagocytosis (Fazekas de St Groth 1948). However, influenza viruses commonly invade the ciliated epithelium of the respiratory tract of the
intact host, which is thought not to be actively phagocytic. Organ cultures of such
epithelium are extremely susceptible to infection (Hoorn & Tyrrell, I969). It was
therefore of interest to use these to investigate the mechanism of entry of influenza
viruses into ciliated epithelial cells; it was uncertain whether the primary target
cells would be the ciliated or the mucus-secreting ceils. As infection was so efficient,
it seemed likely that the virus might 'exploit' in some way the sweeping action of
the cilia and, rather than being moved on by their activity, might attach and then
enter the cells directly or indirectly. Further studies on the entry of virus into
non-ciliated cells appeared during this work and these will be discussed later.
METHODS
Tissue. Organ cultures of guinea-pig trachea were set up in 6o mm. Petri dishes using
the techniques of Hoorn (Hoorn & Tyrrell, ~969), and were maintained at 33 ° overnight in
Eagle's medium in 5 % CO2 in air. Only fragments showing definite ciliary activity next
morning were used in experiments.
Viruses. The influenza strain A 2/ENG 12/64 was used. Inactivated virus was used in the
form of a pool stored at 4 ° with azide for months. It still had full haemagglutinin and neuraminidase activity and it was purified and concentrated first by adsorption and elution from
human red cells, and then by centrifugation to a pellet which was subsequently fractionated
by rate centrifugation in a sucrose density gradient.
Active virus was obtained by harvesting eggs inoculated 2 days before, clarifying the
fluid by low speed centrifugation and producing a pellet by centrifugation at 2o,ooo rev./min.
for 3o min. The pellet was resuspended in Eagle's medium and used with minimum delay.
The titres of haemagglutinin were usually I/IOO,OOO or greater. Active Sendai virus was
prepared in a similar way.
Adsorption of virus. Single pieces of tissue about 2 mm. square were transferred to a cup
in a disposable plastic titration tray (Linbro) in the cold room and each received one drop
of virus. Adsorption usually continued for I hr and then the cup was cut out and filled
with fixative; it was floated first on a bath of warm water if periods of incubation were
required for the experiment.
6
Vlr¢ 9
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78
R. R. D O U R M A S H K I N
AND
D. A. J. T Y R R E L L
Fixation and staining. To obtain good fixation of both membranes and intracellular
structures, two schedules were followed. In the first (sequential fixation) the organ cultures
were flooded with freshly prepared 3 Yo glutaraldehyde in o- I M-phosphate buffer solution.
After 5 rain. they were washed in o.I M-phosphate buffer, and then fixed in I ~o osmic acid
in phosphate buffer (Millonig, 1962 ). They were then washed, dehydrated in alcohol and
embedded in Araldite in the usual way, taking care to ensure adequate infiltration by slow
rotation of the tissues suspended in resin. This procedure gave good fixation of the virus
particles and cilia, but did not reveal much cellular detail. The second schedule was the
double fixation method of Hirsch & Fedorko (t968), using glutaraldehyde and osmic acid
simultaneously. This method, although showing cellular detail of the tracheal epithelium,
resulted in swelling of the ciliary membranes. However, the continuity of the membranes
remained intact whereas they appeared broken in techniques involving the use of bufferedosmic acid alone.
Flat embeddings were used, the epithelium being oriented under a dissecting microscope.
Thin sections were cut and stained with uranyl acetate.
RESULTS
Virus particles were readily identified in sections after exposure to influenza A 2. After
5 rain., most seemed to be entangled in the mucus overlying the cilia. After I hr particles
were floating free, a few were attached to microvilli, but most were seen lined up along
the membranes of cilia, and apparently attached to them (Fig. I). Calculations based
on haemagglutinin titrations suggested that only a small fraction of the virus added was
attached to the epithelium. The number of attached particles of either infectious or noninfectious virus was reduced rapidly to less than 3o % by warming at 37 ° for 3 rain. (Table I).
The effect was seen whether large or moderate numbers of particles were attached. Tissue
was also washed in cold saline after the adsorption step and then incubated as in the preparation of specimens for electron microscopy. About lo units of haemagglutinin was released
into the warmed medium, but a similar amount was also released from washed cultures
held in medium at 4 ° .
The mechanism by which virus particles attached to cold cells was carefully studied. It
seemed that both influenza and Sendal virus often stuck to the membrane by quite a small
area of contact, but occasionally the virus particle or cell profile was altered so that the two
membranes were apposed, sometimes closely, over a larger area. Sometimes the approximated areas seemed rather thickened or blurred, but this was never seen in areas where the
plane of section ran perpendicular to that of the cell membrane, as shown by the fact that
the triple-layered unit membrane was clearly resolved at both sides of the point to which
the virus was attached (Figs. 2, 3).
The membranes of cilia were sometimes interrupted or possibly torn, but it was not
possible to relate this to the adsorption of virus and, after virus had disappeared from
warmed cultures, the membranes were indistinguishable from those of uninfected cultures
(Fig. 4).
In warmed cultures, and mainly in those warmed for 3 rain., profiles were seen which
showed unequivocally (Fig. 5) that the membranes of Sendai virus and the cell fused and
the virus nucleocapsid entered the cilium, became partly uncoiled and apparently spread
down between the membrane and the filaments (Fig. 6). A similar process can apparently
occur when Sendai virus adsorbs to microvilli.
N o such appearances were seen in cultures exposed to influenza virus. Instead, in cultures
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Attachment of" two myxoviruses to ciliated epithelial cells
79
Fig. I. E l e c t r o n m i c r o g r a p h of an organ culture of guinea-pig trachea, i n c u b a t e d w i t h influenza
virus for I hr at 4 °. Large n u m b e r s of virus particles are a d s o r b e d to the cilia. ' D o u b l e f i x a t i o n '
with g l u t a r a l d e h y d e a n d o s m i u m tetro×ide (Methods).
Table
t.
Numbers of influenza virus particles attached to cilia
N u m b e r of particles
r
Times at two
temperatures
C o u n t s / # m . of
ciliary m e m b r a n e
A
C o u n t s as p e r c e n t a g e of those
after I hr of a d s o r p t i o n at 4 °
~
4°
37 °
60
60
6o
60
o
I
3
5
Expt . . . .
z
I
2
3
0"56
-o"13
0"24
0"58
3"20
0"44
o'I5
o'19
o'42
o'88
--
I
I00
-23
42
z
3
100
I00
76
26
29
I3
28
-6-2
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8O
R. R. D O U R M A S H K I N
AND
D. A. J. T Y R R E L L
i!iiilli~;~f !i~i~
Fig. 2. Organ culture incubated with influenza virus for I hr at 4 °. Note appearance of virus
particles, marked by arrows, overlapping the membranes of cilia. Double fixation.
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Attachment o f two myxoviruses to ciliated epithelial cells
Fig. 3. Influenza virus adherent to m e m b r a n e s of cilia, f r o m a n organ culture incubated with
virus for I h r at 4 °. Sequential fixation.
Fig. 4- O r g a n culture incubated with influenza virus for ~ h r at 4 °, t h e n 3 min. at 37 °. Sequential
fixation. Particles adhered to cilia as above; in w a r m e d cultures they were less n u m e r o u s .
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81
82
R . R . D O U R M A S H K I N AND D. A. J. T Y R R E L L
containing active virus, intact virus particles a p p e a r e d to be present within the cilia (Fig. 2).
Serial sections indicated t h a t the a p p e a r a n c e o f virus particles within a cilium, o r crossing
the ciliary m e m b r a n e , always represented a p o r t i o n o f a virus particle, the r e m a i n d e r being
completely outside the cilium. Thus, electron m i c r o g r a p h s o f virus a p p e a r i n g to cross the
Fig. 5. Organ culture incubated with Sendai virus for I hr at 4 °, then 3 rain. at 37 °. Many particles
adhere to the cilia; fusion of virus membrane to membranes of cilia is seen with virus nucleocapsid
distributed along the microtubules of lhe cilia.
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Attachment of two rnyxoviruses to ciliated epithelial cells
Fig. 6. Influenza virus particles adsorbed to microvilli and to the surface o f an epithelial cell. Note
fibres projecting from microvilli. (a) Organ culture incubated for I hr at 4 °. Double fixation.
(b) Organ culture incubated I hr at 4 °, then I min. at 37 °. Sequential fixation.
Fig. 7. Portion of a mucus-secreting cell, from an organ culture incubated with virus for I hr at 4 °,
then ~ rain. at 37 °. Note that the appearance o f the microvilli differs from that o f other epithelial
cells. Sequential fixation.
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83
84
R . R . D O U R M A S H K I N AND D. A. J. TYRRELL
ciliary m e m b r a n e p r o b a b l y represent a superimposition of virus a n d cell m e m b r a n e within
one section.
Virus particles were seen to adsorb to microvilli in the tracheal organ cultures, in the same way
as to cilia (Fig. 6a, b). It may be noted that the filamentous material e m a n a t i n g from the
microvilli, best seen in sequentially fixed cultures, might play a part in virus entrapment.
Fig. 8. Influenza virus adsorbed to human red cells. (a) The cell membrane was sectioned tangentially. Virus particles overlap the edge of tl'te membrane and appear to be fused with it. (b) Cell
membrane sectioned to be visible as a triple layer. There is no evidence of fusion of menabranes of
virus and erythrocyte.
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Attachment of two myxoviruses to ciliated epithelial cells
85
N o virus was seen adsorbed on the microvilli of mucus-secreting cells (Fig. 7).
A n o t h e r a p p r o a c h to the examination o f the relationship o f virus to cell m e m b r a n e was to
study the appearance o f influenza virus particles adsorbed to red blood cells. Virus appeared
to be fused to cell membranes only when the membranes were sectioned tangentially
(Fig. 8 a). In every instance where the double layer of the cell m e m b r a n e was clearly seen,
the virus particles adhered only by means o f their peripheral projections (Fig. 8b). Inactivated and active virus seemed to behave in exactly the same way.
Table 2.
Uptake of influenza virus by Tetrahymena pyriformis
Times at two
temperatures
r
4°
hr
37°
o
I hr
o
1 hr
I hr
I hr
lo rain.
4o rain.
o
I hr
o
I hr
o
i hr
o
i hr
o
Virus haemagglutinin
titre of
Mixture used
supernatant fluid
Virus and Tetrahymena
I [,
Virus only
1/96
Virus and Tetrahymena
1/8
Virus and Tetrahymena
I/, 6
Virus and Tetrahymena
I/2
Virus and Tetrahymena disrupted by blending
i/i28
Virus and Tetrahymena previously incubated
at 33° in cholera filtrate
1/32
Virus and Tetrahymena previously
incubated in broth
1/16
Virus only
I/256
Tetrahymena were sedimented at low speed and mixed with diluted allantoic fluid containing influenza
virus A 2/Eng/344/68. After the indicated procedure and another low speed centrifugation the supernatant
fluids were titrated for haemagglutinating activity.
To study in more detail the relationship between viruses and cilia, some preliminary
experiments were made with a p r o t o z o o n from which the cilia can be recovered in a pure
state.
It was shown first that influenza virus attached rapidly to the ciliate Tetrahymena
pyriformis. A n axenic culture was centrifuged down at low speed and resuspended in
distilled water at approximately IO %. The suspension was mixed with an equal volume o f
allantoic fluid diluted by approximately one-third in distilled water. The results are shown in
Table 2. The haemagglutinin reappeared in the medium after a time and could be recovered by disruption of the organism, but not by treatment with cholera filtrate. On electron
microscopy it became clear that the virus was nowhere attached to cilia, but was ingested
into the p r o t o z o o n ' s food vacuole, from which presumably it was later regurgitated (Fig. 9).
DISCUSSION
It is o f great interest that representative viruses which invade respiratory epithelium can
stick firmly to the cilia; these observations tend to confirm our original hypothesis. It is
desirable now to determine whether viruses o f other groups which also enter the same sort
o f epithelium, attach in the same way.
The mechanism of the entry of Sendai virus nucleocapsid into the cilia is clear and
M o r g a n & H o w e 0 9 6 8 ) have shown that the virus enters chorioallantoic cells in the same
way. We are not convinced from our micrographs that influenza virus does so in the
same way, and only one micrograph of M o r g a n & Rose (I968) actually shows the m o m e n t
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86
R. R. D O U R M A S H K I N
A N D D. A. J. T Y R R E L L
Fig. 9a. Tetrahymenapyriform& incubated with influenza virus; this section shows the cilia of the
buccal overture and virus particles engulfed in the food vacuole (V). Virus particles do not adhere
to cilia.
Fig. 9b. Insert: A higher magnification of the area marked in Fig. 9a.
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Attachment of two myxoviruses to ciliated epithelial cells
87
of penetration; this is unconvincing. There are enormous differences in the electron micrographic appearances of red cells incubated with influenza and Sendai virus. Not only are
cells lysed by exposure to Sendai virus, but nucleocapsid is released; we have confirmed
that this does not occur in influenza virus treatment of red cells. It has been shown that at
least some influenza virus remains accessible to neutralizing antibody for many minutes
after it attaches to chorioallantoic membrane in a way which cannot be reversed by RDE
(Ishida & Ackermann, ~956). After mixing for a few minutes with the normal cytoplasmic
particles which bud from these cells, the virus is fully infectious but seems to be disrupted
when examined by negative contrast (Hoyle, H o r n e & Waterson, t962). We have seen
nothing corresponding to this in virus particles which have interacted with cilia. It is therefore likely that Sendai and influenza viruses react with cells by different mechanisms; in our
view there is no evidence that the nucleocapsid which enters the cell from the Sendai virus
particle is that which initiates infection. The entry of influenza virus into cells has not been
visualized for certain.
During this work, a controversy developed between Morgan and his colleagues, who
demonstrated the entry of myxoviruses into cells by membrane fusion, and Dales and his
colleagues who added to their earlier evidence that viruses such as herpes simplex and
vesicular stomatitis enter cells by engulfment (Dales & Silverberg, ~969; Simpson, Hauser
& Dales, I969; Morgan, Rose & Mednis, ~968).
Viruses of different groups are constructed and multiply in such different ways that it is
to be expected that they also enter cells in different ways. It is important to know which
mechanism of entry relates to each virus, and whether the virus can then initiate infection;
one type of virus may enter different cells in different ways. Thus, influenza viruses have
been seen inside apparent phagocytic vacuoles where they may be digested; in order to
replicate, however, they must penetrate the membrane and enter the cytoplasm and this
process has not been clearly seen. The disagreement between the views of the groups of
Morgan and Dales may be slight, since only herpes simplex virus has been shown to enter
by both membrane fusion and by engulfment, and the experimental systems used differed
appreciably.
We wish to thank Dr G. Schild for supplying inactivated virus and Dr D. Lee for
Tetrahymena pyriformis. We also thank Mrs R. Buckland, Mrs Joan Richmond, Misses
Helen Woodward and Angela Stone, and Mr S. J. A. Tyrrell for technical assistance.
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DOURMASHKIN
A N D D. A. J. T Y R R E L L
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