J. gen. Virol. (1983), 64, 589-598. Printed in Great Britain
589
Key words: influenza virus~pathogenicity~organ cultures~respiratory tract
Distribution of Viral Antigen within the Lower Respiratory Tract of Ferrets
Infected with a Virulent Influenza Virus: Production and Release of Virus
from Corresponding Organ Cultures
By R. H. H U S S E I N I , * C. S W E E T , R. A. B I R D , M. H. C O L L I E AND
H. S M I T H
Department of Microbiology, University o f Birmingham, P.O. Box 363, Birmingham,
B15 2TT, U.K.
(Accepted 8 November 1982)
SUMMARY
Using fluorescent antibody techniques, a semi-quantitative survey has been made of
the distribution of influenza virus antigen in the trachea, main bronchi, and three zones
(hilar, intermediate and alveolar) of all four lung lobes of ferrets following intranasal
inoculation of a virulent clone (7a) of the recombinant influenza virus A/PR/8/34A/England/939/69 (H3N2). The results confirm the indications from our previous
quantitative surveys of infectious virus and histological damage in these areas, namely
that infection is confined largely to airway epithelium and is rare in the alveoli.
Furthermore, in the lung zones, viral antigen resided mainly in the bronchial rather
than bronchiolar epithelium. In attempts to identify the reasons for lack of alveolar
involvement organ cultures of alveolar tissue, from which all major airways had been
removed, produced levels of virus similar to cultures of bronchus and trachea and the
hilar and intermediate lung zones which contain airway and alveolar tissue. Hence, the
lack of alveolar infection in vivo must be due to factors which prevent virus attack of
susceptible alveolar cells. However, these organ culture experiments showed that a
contributing factor could be very poor release of virus from any alveolar cells that do
become infected. In contrast, although cultures of bronchi produced less virus than
those of nasal turbinates (the most susceptible tissue in vivo) they released a high
proportion of their yield and this ease of release may contribute to spread of infection in
vivo.
INTRODUCTION
Human influenza is primarily an upper respiratory tract infection but tracheobronchitis,
bronchiolitis and impaired respiratory function can occur although pneumonia is rare (StuartHarris, 1965; Mulder & Hers, 1972; Douglas, 1975). Respiratory infection in ferrets inoculated
with human strains of influenza virus mimics that in man, predominating in the upper
respiratory tract with some involvement of the lower tract especially for virulent strains
(Matsuyama et al., 1980; Sweet et al., 1981).
The pathogenesis of influenza in the lower respiratory tract (LRT) of the ferret has been
studied in more detail by assessing the distribution in the tract of two virulent (clones 7a and 64c)
and two attenuated (A/PR/8/34 and clone 64d) strains of the recombinant virus A/PR[8/34A/England/939/69 (H3N2) (Sweet et al., 1981). Virus isolations from homogenates of trachea,
the main bronchi, and the hilar, intermediate and alveolar zones of the lung on days 1 to 4
following intranasal inoculation showed that the virulent clones, in contrast to the attenuated
ones, consistently infected the LRT. However, for all infected animals, the virus content of the
hilar zones of the lung lobes was usually higher than in the intermediate zones, while the alveolar
zones were frequently free of virus. This suggested that influenza virus replicated mainly in
areas where bronchial and bronchiolar epithelium predominated. Quantitative estimation of
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590
R. H. HUSSEINI AND OTHERS
histological damage in the various areas supported this view; lung damage was minimal, but it
was localized in the airways and not in the alveoli (Sweet et al., 1981).
The presence of virus in homogenates of any region of the respiratory tract does not
necessarily mean that replication occurred there, because virus can be translocated by
mucociliary action or drainage. Similarly, histological damage may occasionally occur at sites
distal to viral replication. To be sure of the pattern of infection, it was necessary to visualize viral
antigen in the trachea and main bronchi and within the different regions of the lung. Here, we
describe first a semi-quantitative survey of the distribution of viral antigen detected by
fluorescent antibody within the LRT of ferrets 1 to 4 days after inoculation of the virulent clone
7a.
This survey confirmed the conclusions from previous work which raise an important
question. Why does influenza virus attack the epithelial lining of the airways but not alveolar
tissue ? Previous work suggested a possible explanation. Comparison of virus yields from organ
cultures of nasal turbinates and lung tissue taken from a mixture of the intermediate and alveolar
zones as defined by Sweet et al. (1981), showed that the turbinates produced about tenfold more
virus per cell than the lung tissue and released a much greater proportion (about 30%) of the
virus yield than did lung cultures (about 6%); observations with fluorescent antibody showed
that ciliated cells were the predominant cell type infected in the turbinate cultures, and alveolar
type I and type II cells in the lung cultures (Kingsman et al., 1977a, b; Cavanagh et al., 1979;
Matsuyama et al., 1980). Hence, one factor contributing to the higher susceptibility of nasal
mucosa compared with lung tissue appeared to be a higher capacity of the ciliated cells of the
former to produce and release influenza virus compared with the alveolar cells of the latter.
Could a similar explanation apply to the differences in susceptibility in LRT? Clearly, the
virtual absence of alveolar infection in vivo is not explained by the organ culture data since some
virus production occurred in alveolar cells. However, the greater susceptibility of airway
epithelium may, like that of nasal turbinates, reflect a high capacity to produce and release virus.
Hence virus production and release and median infectious doses of clone 7a have been examined
in organ cultures of ferret nasal turbinates, trachea, main bronchi and the hilar, intermediate
and alveolar zones of the lung (Sweet et al., 1981). With regard to the alveolar zone, care was
taken to exclude bronchi and large bronchioles from the organ culture pieces so as to obtain a
better estimate of virus production and release from alveolar tissue than had been achieved
previously when the tissue pieces contained some large bronchioles and even small bronchi
(Kingsman et al., 1977a, b; Cavanagh et al., 1979; Matsuyama et al., 1980).
METHODS
Virus. Clone 7a (H3N2) of the recombinant influenza virus A/PR/8/34-A/England/939/69 was described
previously, together with its assay and the preparation of seed and working stocks (Sweet et al., 1974).
Intranasal inoculation offerrets. Ferrets were inoculated with 106 EBIDso (50% egg-bit infectious doses) of clone
7a as described by Toms et al. (1976) and infection checked by showing that nasal washes (Toms et al., 1976) taken
24 h post-inoculation contained > 105.0 EBIDs0 virus/ml.
Preparation o f respiratory tract tissue for sectioning. Respiratory tissue was removed from freshly killed ferrets 1 to
4 days post-inoculation (two ferrets/day) and divided into trachea, main bronchi and the hilar, intermediate and
alveolar zones of all four major lung lobes as described previously (Sweet et al., 1981). Tissues were fixed
immediately in cold 95 % (v/v) ethanol for 24 h, transferred to cold absolute ethanol for a further 48 h, embedded in
Ralwax (Raymond Lamb, London, U.K.) and stored at - 20 °C prior to sectioning.
Preparation o f antiserum against clone 7a. Virus, grown in the chorioallantoic membrane of 10 to 12-day-old
embryonated hens' eggs for 48 h at 35 °C, was harvested, concentrated about 20-fold with polyethylene glycol 6000
(Hopkins and Williams, U.K.) and centrifuged at 12000g for 90 min at 4 °C. The pellet was dialysed against
phosphate-buffered saline (PBS) overnight. Rabbit antiserum was produced by intraperitoneal injection of
4 × 104 HA (haemagglutination) units of virus on days 0, 28 and 35, the rabbits being bled at day 41. Further
batches of antiserum were raised at irregular intervals ( > 4 weeks) in the same rabbits by intravenous inoculation
of similar doses of virus and bleeding 6 to 8 days later. The sera were treated with NazSO4 (16%, w/v), centrifuged
at 12000g (30 rain, 25 °C) and the pellet dialysed overnight against FA phosphate buffer ('FA PBS'; NaC1, 8.5;
Na2HPO4, 1.06; NaH2PO 4 . 2H20, 0-39 g/l; pH 7.1). IgG fractions, obtained by gel filtration on Sephadex G200
and collected in FA PBS, contained from 2560 to 5120 haemagglutination-inhibition (HI) units/ml (Sweet et al.,
1974).
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Influenza virus pathogenicity
591
lmmunofluorescent examination of sections. Sections (2 gm), cut from the Ralwax-embedded specimens, were
stained with a 1/20 dilution of Bacto FA Rhodamine counterstain (Difco) for 1 h in a humidified chamber at room
temperature. They were washed with FA PBS and covered with a 1/4 dilution of the rabbit anti-influenza IgG for
1 h. Sections were washed again in FA PBS, stained with a 1/16 dilution of fluorescein-labelled sheep anti-rabbit
IgG (Wellcome Reagents, Beckenham, U K . ) for 30 min, washed, dried and mounted in alkaline-buffered
glycerol (NaHCO3, 0.72g; NazCO3, 0.16g; distilled water, 100ml; glycerol, 900ml). Sections were then
examined with a Photo-Microscope 3 [Carl Zeiss (Oberkochen) Ltd, London, U.K.] using epi-illumination.
Suitable control experiments with (i) infected tissue and normal rabbit antiserum, and (ii) uninfected tissue and
the rabbit influenza IgG, ensured that the fluorescence observed in the experimental samples was specific.
Estimation of viral antigen in trachea and main bronchi usingfluorescein-labelled antibody. The trachea and main
bronchi were sectioned longitudinally so that a large surface area of the epithelial lining could be seen in each
section. Two to four sections (10 to 15 fields each) were examined for each tissue of each animal.
Enumeration of bronchi, bronchiolesand alveoli containing viral antigen in the hilar, intermediate and alveolar zones of
the lung usingfluorescein-labelled antibody. Sections were taken throughout the three zones (Sweet et al., 1981) of
each of the four lobes of the lung of each animal. Accurate quantification was impossible because of the irregular
shapes of the sections and the large variation in size of the lung lobes within an animal and between animals.
Nevertheless, an attempt was made to survey similar areas of tissue in each zone of all lung lobes of each animal.
For each lung lobe, 16 to 20 sections (10 to 15 fields/section) from alveolar zones (which were smaller than the other
two zones) were examined. Six to eight sections (30 to 40 fields/section) from the intermediate zones and four to
eight sections (30 to 40 fields/section) from the hilar zones were also examined. The total number of bronchi
(defined as airways totally or partially surrounded by mural cartilage) and bronchioles (not surrounded by
cartilage) transected were counted, as were the number showing specific fluorescence in the epithelial linings.
The results were expressed as the percentage of bronchi or bronchioles showing specific fluorescence. In the
alveolar tissue of all zones, fluorescing cells were seen only occasionally; these were counted and expressed as the
number of fluorescing cells per section.
Quantification offluorescence in the epithelia of the airways. In addition to the enumeration of the airways showing
fluorescence as described above, an attempt was made to quantify the extent of fluorescence in the epithelium in
the following manner. In the estimation of the sections of trachea, main bronchi and bronchi and bronchioles in
the three lung zones as described above, the number of fluorescing cells in the epithelium surrounding each
airway was scored as ' + ' (< 10~ of the epithelial cells fluorescing), ' + + ' (10 to 50~0 fluorescing), or ' + + + '
(> 50~ fluorescing). The results were recorded as the percentages of the total fluorescing airways exhibiting
(a)
.
, - ,. -
Trachea
~ ~ C / I
--
,!i/f;il
} iii
ioilCh i
.ronc ,o,e
(b)
Bronchus~.
o~..,"o o o ~.:~
Alveoli
x..~_- o_ _ _ _ ~ . ~ . . /
Alveolar zone
Intermediate zone
Intermediate & outer
(alveolar) zones
Discarded pleural
membrane
Fig. 1, Regions of the lower respiratory tract, examined in organ culture (a). Dorso-ventral slices (1 to
2 ram) of tissue from intermediate and alveolar zones were removed and further dissected as shown in
(b).
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592
R. H . H U S S E I N I
AND
OTHERS
fluorescence at the three arbitrary chosen levels. This method of assessment did not take into account the
differences in diameter of the airway which for the bronchi were much larger in the hilar region than in the
intermediate and outer regions.
Organ cultures. Nasal turbinates, trachea, main bronchi and hilar, intermediate and alveolar zones of the lung
were excised as shown in Fig. 1. Small pieces were prepared for organ culture as described previously (Kingsman et
al., 1977a; Cavanagh et al., 1979) and inoculated as soon as possible after removal from the animal ('fresh'
cultures; see Kingsman et al., 1977a). In some experiments (expt. 4 and 5 in Table 4) the trachea and main bronchi
were cut into rings (two per Petri dish)rather than pieces in an attempt to preserve the fragile epithelial lining. To
obtain sufficient nasal turbinate, tracheal and bronchial tissue for replicate organ cultures, tissue from two ferrets
was pooled. The pieces of the different tissues varied in size but attempts were made to ensure that the total
epithelial areas exposed to virus were approximately similar. The Petri dishes containing the inoculated tissue were
enclosed in airtight containers, gassed with 5 % CO2/95 % air and completely immersed in a water bath at 39 °C to
simulate the temperature of the lungs of adult ferrets (Belshe et al., 1978). Virus yields in the culture medium and in
homogenates of the tissue were assayed 24 h after inoculation. At the same time some tissue pieces inoculated with
106.7 EBIDs0 of clone 7a were sectioned and examined for infected cells using fluorescein-labelled antibody as
described above. 50% median infectious dose (MIDs0) determinations were made as described previously
(Kingsman et at., 1977b) except that 10 organ culture dishes were used for each dilution and yield was assessed in
the tissue pieces as well as the culture medium.
RESULTS
Fluorescence in the trachea and main bronchi
Fluorescence in the epithelial lining of the t r a c h e a and m a i n b r o n c h i was e v i d e n t in all
sections t h r o u g h o u t days 1 to 4 post-inoculation (Fig. 2 a and b r e s p e c t i v e l y ; T a b l e 1). I n b o t h
regions fluorescence was m a x i m a l on days 2 a n d 3, decreasing by day 4. T h e e p i t h e l i u m o f the
m a i n b r o n c h i t e n d e d to show m o r e fluorescence t h a n that o f the t r a c h e a , but in both, t h e
fluorescence r e m a i n e d focal and did n o t e n c o m p a s s the e n t i r e e p i t h e l i u m . M u c o u s glands
a r o u n d the airways fluoresced in some sections (Fig. 2a) but this could h a v e b e e n non-specific
since these glands occasionally fluoresced w h e n treated w i t h n o r m a l r a b b i t s e r u m instead o f
anti-7a serum.
Enumeration of bronchL bronchioles and alveolar cells fluorescing in the three lung regions
Fluorescence was o b s e r v e d in b r o n c h i and bronchioles in all zones o f the lung (Fig. 2 c, d a n d
e); fluorescing debris was occasionally seen in their l u m i n a on days 3 and 4 p o s t - i n o c u l a t i o n (Fig.
2c).
T a b l e 2 s u m m a r i z e s the results o f counts o f total and fluorescing b r o n c h i and b r o n c h i o l e s a n d
• the occasional fluorescing cell in the alveolar r e g i o n f r o m the three lung zones o f all four lobes o f
two ferrets killed on days 1, 2, 3 and 4 post-inoculation. T h e results f r o m all four lobes are
c o m b i n e d for e a c h animal. A l t h o u g h the figures v a r i e d f r o m lobe to lobe [e.g. the p o s i t i v e
T a b l e 1. Semi-quantitative assessment o f fluorescence in trachea and main bronchi of ferrets infected
with clone 7a
Day after
inoculation
1
2
3
4
•
Ferret no.
1
2
3
4
5
6
7
8
Level of fluorescence* in
~'
Trachea
Main bronchi
++
++
+
+
++
+++
++
++
++
++
+
++
+
+
+
++
* Two to four sections (10 to 15 fields/section) were viewed from both trachea and main bronchi of each animal;
all sections showed some fluorescence. The levels were: +, < 10 ~ of the epithelial cells fluorescing; + +, 10 to
50~ fluorescing; + + +, > 50~ fluorescing.
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Influenza virus pathogenicity
593
@
m
O
Fig. 2. Fluorescence in cells of different regions of the lower respiratory tract of ferrets infected with
clone 7a (3 days post-inoculation). (a) Trachea, x 80 [also note fluorescing mucous glands (MG) and
basement membrane (BM) showing non-specific fluorescence due to rhodamine counter stain]; (b)
main bronchus, × 125 (BM fluorescing non-specifically); (c) bronchus in hilar region, × 125 (note
fluorescing debris in lumen and BM fluorescing non-specifically); (d) bronchiole in intermediate
region, x 125 (note negative adjacent bronchiole); (e) bronchiole in intermediate region, x 125 (only
slight fluorescence); (jr) alveolar cell in alveolar zone, × 200.
bronchi in the intermediate zone varied from 5 3 ~ (lobe 2) to 97~o (lobe 3) in one animal, while
the positive bronchioles in the same region varied from 1 ~ (lobe4) to 9 ~o (lobe 1)], this variation
did not affect the overall pattern of infection within the lung which is indicated by the figures in
Table 2.
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594
R.H.
HUSSEINI A N D OTHERS
Table 2. Enumeration of bronchL bronchioles and alveolar cells containing fluorescence in the three
lung zones of ferrets infected with clone 7a*
Fluorescing alveolar
cells
g
Bronchi
Bronchioles
Total no.
Lung
Day a~er
Ferret ,
~
~r - ~ - - ~
No. per of sections
region
inoculation
no.
Total ~Fluorescing Total ~ Fluorescing section examined
Hilar
1
1
67
45
544
1
1-8
24
2
44
48
363
0
0
16
2
3
92
90
212
1
0.4
24
4
94
99
584
3.
0.2
24
3
5
54
93
169
2
0.9
27
6
104
100
539
6
0.4
24
4
7
41
90
350
10
1.1
36
8
124
90
519
6
0.6
23
Intermediate
1
1
203
47
1233
0-4
2.9
32
2
111
30
502
0
0
24
2
3
212
72
1028
2
1.0
34
4
91
97
1301
3
0.6
40
3
5
73
90
368
4
1.8
38
6
60
97
1272
4
0.6
32
4
7
109
65
688
7
3.1
37
8
175
81
1307
4
0.7
32
Alveolar
1
1
5
0
996
0
0.5
64
2
0
0
382
0
0
27
2
3
58
16
584
1
1.2
70
4
0
0
745
0
0.1
66
3
5
0
0
128
12
1-0
67
6
0
0
903
1
0.2
64
4
7
3
0
359
3
I-7
64
8
14
86
1392
3
0.4
64
* For each of the four lung lobes of each animal, 4 to 8 sections (30 to 40 fields/section) from the hilar, 6 to 10
sections (30 to 40 fields/section)from the intermediate, and 16 to 20 sections (10 to 15 fields/section)from the outer
regions were examined. The results from the four lobes were combined to give the figures shown. For the
distinction between bronchi and bronchioles, see text.
As early as 1 day post-inoculation, approximately 50 ~ of the bronchi transected in the hilar
zone exhibited fluorescence. By days 2 to 4 post-inoculation, 90 to 1 0 0 ~ of the bronchi were
fluorescing. A similar pattern was seen in the bronchi of the intermediate zone except that the
percentage of airways positive tended to be lower. The alveolar zone did not usually contain
bronchi but when present they were often fluorescing.
With regard to the bronchioles, only a small proportion showed fluorescence on any day in any
region. Fluorescing bronchioles in the hilar zone increased gradually from about 1~ on day 1 to
6 to 1 0 ~ by day 4 post-inoculation. A similar pattern was seen in both the intermediate and
outer zones except for the large figure (12~o) for one ferret on day 3 post-inoculation. The few
fluorescing bronchioles were mainly of the larger size while almost all terminal and respiratory
bronchioles (Junqueira & Carneiro, 1980) were negative.
Occasionally, fluorescing cells could be seen in the alveolar region (Fig. 2 f ) but the n u m b e r s
were always low and did not exceed 3 cells per section even on days 3 and 4 post-inoculation
(Table 2). Often, fluorescing cells in the alveolar region could not be seen in sections (see areas
surrounding the bronchi and bronchioles in Fig. 2). The exact nature of the few fluorescing cells
in the alveolar region (pneumonocytes or macrophages) could not be determined.
Semi-quantitative assessment of the level of fluorescence in the bronchi and bronchioles of the
three lung zones
The levels of fluorescence in the positive bronchi and bronchioles in the different zones of the
lung are shown in Table 3. While Fig. 2(c) and (d) depicts a + + + ( > 5 0 ~ ) fluorescence, Fig.
2(e) depicts a + ( < 10~) fluorescence.
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Influenza virus pathogenicity
595
Table 3. Semi-quantitative assessment of the level offluorescence in the positive bronchi and bronchi-
oles in the three lung zones of ferrets infected with clone 7a
Lung
region
Hilar
Day a~er
inoculation
1
2
3
4
Intermediate
1
2
3
4
Alveolar
1
2
3
4
Ferret
no.
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
% Bronchi* fluorescing
~ Bronchioles* fluorescing
at levelt
at levelt
~
A
~ r
~
+
++
+++
+
++
+++
70
30
0
100
0
0
67
33
0
0
0
0
23
49
28
100
0
0
9
81
10
88
12
0
2
12
86
50
38
12
3
63
34
53
43
4
22
60
18
6l
39
0
12
51
37
80
20
0
68
32
0
80
20
0
73
27
0
0
0
0
34
51
14
83
17
0
23
69
8
91
9
0
8
26
66
85
15
0
14
62
24
57
41
3
62
27
11
84
16
0
20
68
12
90
10
0
0
0
100
0
0
0
0
75
0
0
0
0
0
0
0
0
0
0
0
25
0
0
0
0
0
0
0
0
100
0
40
38
100
76
0
0
0
40
62
0
24
0
0
0
20
0
0
0
* The distinction between bronchi and bronchioles, the number of fields viewed and sections examined for the
four lung lobesof each animal are outlined in footnotes to Table 2. Only the bronchi and bronchioles showing some
fluorescence (see Table 2) were included.
t Three arbitrary levels were chosen: +, < 10% of the epithelial cells fluorescing; + +, 10 to 50 ~ fluorescing;
+ + +, >50~ fluorescing.
On the first day after inoculation, most (about 7 0 ~ ) of the positive bronchi in both the hilar
and intermediate zones showed < 1 0 ~ fluorescence, indicating that although m a n y bronchi
were infected (about 50 ~ ; Table 2) they were not heavily so. By 2 days post-inoculation, the
n u m b e r of cells fluorescing in the bronchi of both the hilar and intermediate zones increased,
reaching a m a x i m u m on day 3 (86 to 9 8 ~ of the airways of both zones had over 10~o of their
epithelial cells fluorescing; 24 to 8 6 ~ had > 50~o fluorescing) and declined on day 4 (38 to 8 8 ~
of airways with > 1 0 ~ cells fluorescing; 11 to 3 7 ~ had > 509/00fluorescing). Similarly, most of
the relatively few bronchioles which showed fluorescence in both zones (Table 2) contained
< 1 0 ~ fluorescing cells on days 1 and 2 post-inoculation and although more cells became
involved on days 3 and 4 post-inoculation, the level of fluorescence in more t h a n half of the
bronchioles remained at less than 10~. Bronchi and bronchioles of the hilar region tended to
show more cells fluorescing than those in the intermediate zone at days 2, 3 and 4 postinoculation. In addition, the diameters of the bronchi in the hilar zone tended to be greater than
those of the bronchi in the intermediate zone thus emphasizing the greater fluorescence in the
hilar region.
In the alveolar zone, bronchi occurred only rarely and showed relatively few fluorescent cells.
In the relatively few positive bronchioles (Table 2) the level of fluorescence only reached the
+ + level in an appreciable n u m b e r on day 3 post-inoculation.
Production and release of virus from organ cultures of respiratory tissues
Nasal turbinate organ cultures produced far more virus than the other respiratory tissues
(Table 4) and the MID50 for these turbinate cultures was low [0.8 + 0.1 (standard error)
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596
R.H.
HUSSEINI AND OTHERS
Table 4. Production and release of influenza virus clone 7a by fresh cultures of ferret respiratory
tissue at 39 °C
Tissue
Nasal turbinates
Trachea
Main bronchi
Hilar zone
Intermediate zone
Alveolar zone
Experiment
no.*
1
2
3
1
3
4
5
1
3
4
5
1
2
3
1
2
3
1
2
3
Total virus yieldt
(loglo EBIDso)
6-4
5-7
6-0
3-9
3.0
4.2
3-5
4.5
3.8
4.1
3-7
4.4
3.7
5.2
4.3
4.4
5.3
4.4
4.2
5.1
(0-1)
(0.1)
(0-2)
(0-1)
(0.1)
(0-1)
(0-2)
(0.2)
(0-2)
(0-1)
(0-2)
(0-2)
(0.2)
(0-1)
(0-2)
(0.1)
(0.2)
(0.2)
(0.2)
(0.1)
Virus yield in
culture medium
( ~ of total yield)
16 (5)
23 (4)
25 (7)
78 (4)
56 (5)
52 (9)
42 (6)
70 (9)
63 (5)
36 (8)
23 (4)
19 (7)
17 (5)
14 (4)
27 (7)
15 (4)
9 (3)
0.4 (0.3)
6-0 (1.0)
0.5 (0.2)
* In experiments 1 and 3 small pieces of trachea and bronchi were cultured as for other tissues, in experiments 4
and 5 tracheal and bronchial rings (2/dish) were used.
~"Organ cultures were prepared and inoculated (4-7 logloEBIDs0/culture) as described in Methods. Figures
represent the mean of yields from 6 replicate cultures (standard error in parentheses) at 24 h after infection.
Appropriate controls showed that <0.5 log~oEBIDs0 remained after 24 h.
log10 EBIDs0]. Total virus production was similar for all three zones of the lung (Table 4) as were
their MIDs0s (2.3 + 0.1, 2.4 + 0.1 and 2.1 + 0-1 logl0EBID50 for hilar, intermediate and
alveolar zones respectively). Virus production by organ cultures of the main bronchi (Table 4)
was similar to, but possibly lower than, that of the three zones and the MIDso was higher
(3.3 + 0-3 lOgl0EBIDs0). Tracheal cultures also showed a tendency to produce lower yields and
had an MIDso of 2.9 + 0.4 log10 EBIDso. Release of virus into the culture medium was highest
for bronchial and its tracheal cultures especially when they were cultured in small pieces (Table
4). The lower release with the ring-type cultures (expt. 4 and 5, Table 4) may relate to less
disturbance of the epithelial layer in such cultures. Release from nasal turbinate cultures (16 to
25 %) was similar to that observed in previous work (Cavanagh et al., 1979). The striking result
was the very poor release of virus from cultures of the alveolar zone from which bronchial and
bronchiolar epithelium had been largely excluded (Table 4). Release by cultures of the hilar and
intermediate zones reflected the fact that they were mixtures of airway epithelium and alveolar
cells.
Fluorescein-labelled antibody showed that the infected cells in all cultures were near the
periphery of the tissue pieces (compare Kingsman et al., 1977 a). In nasal turbinate, tracheal and
bronchial cultures, epithelial cells were infected but the foci of infected cells were more sparsely
distributed in the trachea and bronchi than in the turbinates. In cultures of all three lung zones,
appreciable numbers of isolated alveolar cells fluoresced and, when they were present at the
periphery of the organ culture piece, the epithelium of bronchi and bronchioles also fluoresced.
The latter fluorescence was seen often in cultures of the hilar and intermediate zones but rarely
in those of the alveolar zone.
DISCUSSION
This semi-quantitative assessment of the distribution of viral antigen in various areas of the
lower respiratory tract has confirmed the indications from previous quantitative surveys of
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Influenza virus pathogenicity
597
infectious virus and histological damage (Sweet et al., 1981) and provided additional
information. Firstly, fluorescing cells were not seen in significant numbers in the alveolar tissue
of any of the three lung zones during all 4 days of infection (Tables 2 and 3); this confirms the
main suggestion of Sweet et al. (1981) that, in the ferret at least, infection of lung tissue is
bronchial and bronchiolar and not alveolar. Secondly, the previous studies had indicated that
bronchial and bronchiolar epithelium were the susceptible areas for infection but did not
distinguish between the two; clearly, however, the bronchial epithelium is preferred (Tables 2
and 3), at least for clone 7a. Thirdly, virus found in the trachea and main bronchi could not have
been derived solely from translocation of virus from other parts of the respiratory tract (Sweet et
al., 1981) since viral antigen was found in the epithelial layer of both organs on the first and later
days post-inoculation (Fig. 2a, b; and Table 2). Finally, the progressive pattern of fluorescence
found on days 1, 2, 3 and 4 in the trachea, main bronchi and the three lung zones indicate a
descending spread of infection. Thus, fluorescence was substantial in the trachea and main
bronchi on days 1 and 2 after inoculation (Table 1) but only became so on days 2 and 3 for the
bronchi in the hilar and intermediate zones and on days 3 and 4 for the few bronchioles that
became infected in these zones (Tables 2 and 3). A similar descending spread of infection had
occurred in ferret neonates (Collie et al., 1980) but in mouse infection with adapted strains
ascending (Hers et al., 1962; Albrecht et al., 1963), descending (Nayak et al., 1979; Yilma et al.,
1979) and simultaneous infection of all parts (Frankova, 1975) took place according to strain and
method of inoculation.
One possible reason for the lack of alveolar involvement is poor production and release of
virus by alveolar cells compared with airway epithelial cells; this has been investigated using
organ cultures of the appropriate tissues. Total virus yields from alveolar tissue from which
major airways had been excluded were as high as those from the other lung zones, which
contained appreciable amounts of bronchial and bronchiolar epithelium, and possibly higher
than those of bronchial and tracheal cultures where alveolar cells were absent. Hence, the
absence of alveolar infection in vivo is due to factors which prevent virus attacking susceptible
alveolar cells such as poor virus seeding of distal regions of the tract after intranasal inoculation,
clearance of virus by mucociliary action and destruction of virus and virus-infected ceils by
alveolar macrophages. However, poor virus release from the few cells that do become infected
could be a contributing factor to the continued low involvement of alveolar cells. This poor
release was indicated not only by the relative virus yields from medium and tissue pieces of
organ cultures but also by the demonstration, with fluorescent antibody, that infected alveolar
cells were found singly or in small groups at the periphery of tissue pieces in contrast to the large
groups of epithelial cells seen in infected airways.
Virus production by organ cultures of bronchus, the epithelium of which, both external and
internal to the lung, is predominantly involved in LRT infection was not as high as that from
nasal turbinates, the most susceptible tissue in vivo. In addition, bronchial organ cultures had a
higher MIDs0 than nasal turbinates. However, the percentage release of virus from bronchial
organ cultures was higher than that from nasal turbinate cultures and much higher than that
from alveolar tissue. This ease of release of virus from the bronchial epithelial cells should
enhance spread of infection and observations with fluorescent antibody on the dissemination of
infection within the epithelium of infected airways support this view.
The reason for the predominance of bronchial over bronchiolar infection is unknown. The
relatively small involvement of the bronchioles and the slow descending nature of the infection
suggests that mucociliary action limits the spread of virus down the respiratory tree; in mice at
least this action appeared relatively unimpaired by virus attack (Green et al., 1977). However,
more specific mechanisms may operate such as a greater susceptibility of the epithelial and/or
goblet cells of the bronchi to infection with influenza virus compared with those in the
bronchioles.
This workwas supportedby the MedicalResearchCouncil. We gratefullyacknowledgethe technical assistance
of Mrs J. Gem, Mrs S. Chalder and Mrs L. Harper.
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598
R. H. H U S S E I N I A N D O T H E R S
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