Download Virulent Avian Influenza A Viruses

J. gen. Virol. (1989), 70, 2887-2895.
Printed in Great Britain
2887
Key words: avian influenza virus/lyrnphocytes/macrophage
Virulent Avian Influenza A Viruses: Their Effect on Avian Lymphocytes
and Macrophages in vivo and in vitro
By H A N A V A N C A M P E N , * B E R N A R D C. E A S T E R D A Y AND
V I R G I N I A S. H I N S H A W
Department of Pathobiological Sciences, School of Veterinary Medicine, University of WisconsinMadison, 2015 Linden Drive West, Madison, Wisconsin 53706, U.S.A.
(Accepted 9 July 1989)
SUMMARY
To investigate the pathogenesis of virulent avian influenza A viruses, the effect of
A/turkey/Ont/7732/66 (H5N9) (Ty/Ont), A/tern/South Africa/1961 (H5N3)
(Tern/S.A.) and A/chicken/Pennsylvania/1370/83 (H5N2) (Ck/Penn) on avian
lymphoid cell populations was examined in vivo. Previous studies have shown that
infection of chickens with Ty/Ont resulted in the extensive destruction of lymphoid
tissues. In this study, other virulent avian H5 influenza viruses, Tern/S.A. or Ck/Penn,
had little or no effect on lymphoid tissues of infected chickens. Therefore the effect of
Ty/Ont on lymphoid tissue is a specific activity of this virus only and not of other
virulent avian H5 influenza strains. To examine the role of viral replication in the
destruction of lymphocytes, in vitro cultures of avian macrophages and lymphocytes
were inoculated with Ty/Ont. Macrophages supported the synthesis of viral proteins
whereas lymphocytes produced small, but detectable amounts of viral protein;
however, infectious virus was not produced by either cell type. Furthermore
inoculation of chicken spleen cells with Ty/Ont in vivo and in vitro had a profound effect
on the proliferative response of lymphocytes to concanavalin A. These results suggest
that Ty/Ont infects macrophages as well as lymphocytes in the chicken, and the effects
of the virus on both cell types may well contribute to lymphoid necrosis.
INTRODUCTION
Virulent strains of avian influenza A viruses have caused severe losses to the poultry industry
both in mortality and in costs of eradication (Kawaoka et al., 1984). The mechanisms by which
these virulent strains cause disease and death of their hosts is not clear. In particular, the specific
cells involved in viral replication and the mechanisms by which these viruses injure these cells
have not been defined. Previous in vivo studies indicated that the virulent avian influenza virus,
A/turkey/Ont/7732/66 (H5N9) (Ty/Ont) has a pronounced and specific effect on the lymphoid
cell populations of its avian host (Van Campen et al., 1989). Inoculation of chickens with Ty/Ont
resulted in the destruction of lymphocytes in both blood and tissues which was accompanied by
high viral titres in these same tissues. Viral antigen was found in discrete, round cells within
areas of lymphoid necrosis, suggesting that Ty/Ont replicates in cells present in lymphoid tissues
and that viral replication is related to tissue destruction.
The first portion of this study was undertaken to determine whether the destructive effect of
Ty/Ont on lymphoid tissues was also a characteristic of other virulent avian H5 influenza A
viruses. In previous reports describing the histopathological changes in chickens infected with
virulent influenza A viruses, foci of lymphoid necrosis in the spleen were found in a small
proportion of chickens inoculated with A/tern/South Africa/1961 (Tern/S.A.) (Uys & Becker,
1967), whereas necrosis of lymphoid tissue was not characteristic of A/chicken/Pennsylvania/1370/83 (Ck/Penn) infection (Acland et al., 1984). Specific cell populations involved in
0000-9006 © 1989 SGM
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H. VAN CAMPEN, B. C. EASTERDAY AND V. S. HINSHAW
t h e r e p l i c a t i o n o f t h e s e v i r u l e n t s t r a i n s o f i n f l u e n z a v i r u s w e r e n o t i d e n t i f i e d in e i t h e r case. I n
t h i s study, l y m p h o i d tissues f r o m c h i c k e n s i n o c u l a t e d w i t h T e r n / S . A . or C k / P e n n w e r e
e x a m i n e d for t h e p r e s e n c e o f v i r a l a n t i g e n .
T h e effects o f T y / O n t o n l y m p h o i d cell p o p u l a t i o n s were e x a m i n e d in vitro to d e t e r m i n e
w h e t h e r v i r a l r e p l i c a t i o n w a s i n v o l v e d in t h e i r d e s t r u c t i o n . S p l e n i c l y m p h o c y t e a n d a d h e r e n t
cell c u l t u r e s i n o c u l a t e d in vitro w e r e a s s a y e d for t h e p r o d u c t i o n o f v i r a l p r o t e i n s a n d i n f e c t i o u s
virus. F u r t h e r m o r e , t h e effects o f T y / O n t o n t h e p r o l i f e r a t i v e r e s p o n s e o f l y m p h o c y t e s w e r e
d e t e r m i n e d . T h e results suggest t h a t t h e n e c r o s i s o f l y m p h o i d tissues o b s e r v e d in vivo m a y result
f r o m i n f e c t i o n o f m a c r o p h a g e s as well as l y m p h o c y t e s , b y T y / O n t .
METHODS
Chickens. White Leghorn chickens were obtained from a closed flock serologically negative for influenza and
Newcastle disease virus.
Viruses. The viruses were obtained from the virus repository at the School of Veterinary Medicine, University of
Wisconsin-Madison. All viruses were grown in embryonated chicken eggs; after 2 days at 35 °C, allantoic fluid
was harvested and stored at - 70 °C. The virus, Ty/Ont, was from the first egg passage of virus isolated from the
lung of an infected turkey. The virus, Ck/Penn, was kindly provided by J. Pearson, National Veterinary Services
Laboratory, Ames, Iowa, U.S.A. The studies with Ck/Penn were conducted in a P3 containment laboratory at the
School of Veterinary Medicine, University of Wisconsin-Madison.
Infection study. Ten chickens were inoculated with l03 EIDso of Tern/S.A. and four chickens were inoculated
with 107.5 EIDs0 of Ck/Penn, intratracheally. The chickens were killed at 12 h (Tern/S.A.) or 24 h (Ck/Penn)
intervals, necropsied and examined for gross lesions. Samples of trachea, thymus, skeletal muscle, spleen, liver,
pancreas, duodenum, caecum, bursa, kidney, lung and air sac, heart, brain, and bone marrow were collected and
fixed in 10~ buffered formalin for standard histopathological examination and for immunoperoxidase staining.
Tissues placed into 10~ buffered formalin were processed and embedded in paraffin within 48 h of collection.
Sections were stained with haematoxylin and eosin for examination by light microscopy or stained using the
immunoperoxidase technique described below.
Cellpreparation and in vitro infection ofcetls. Chickens were killed by cervical dislocation and their spleens were
aseptically removed and placed in a plastic Petri dish containing minimal essential medium (MEM) (Gibco)
supplemented with 10~ bovine serum albumin (BSA), penicillin (100 units/ml), streptomycin.(100 ~tg/ml) and
2 mM-L-glutamine. The capsule was removed from the spleen and the minced tissue was passed through a tissue
sieve (Bellco Glass).
For preparation of lymphocytes, 4 ml aliquots of the resulting cell suspension were centrifuged at 60g for 15 min
in 12 x 75 mm polypropylene tubes (Becton-Dickinson). The supernatants containing > 9 5 ~ lymphocytes were
collected and the ceils were washed twice by centrifugation at 400 g for 10 min and resuspension in fresh media. To
remove adherent cells, the suspension was passed through a sterile nylon wool column, incubated for 1 h at 39.5 °C
and eluted with prewarmed medium (Schaefer et al., 1985). Spleen cells used for lymphocyte proliferation assays
were obtained in the same manner except that adherent cells were not removed by passage over the nylon wool
column.
Cultures were enriched for adherent cells by allowing the spleen cell suspensions to adhere to 100 x 20 mm
plastic Petri dishes (Becton-Dickinson) or two-chambered slides (Miles Laboratories) for 4 h at 39-5 °C. Nonadherent cells were removed by vigorous washing with prewarmed medium and the remaining adherent cells were
cultured with fresh medium at 39.5 °C.
Cultures of lymphocytes were inoculated with 0.1 or 10 EIDso/cell of Ty/Ont in 15 ml polypropylene centrifuge
tubes (Costar) for 30 min. Cells were washed twice in medium by centrifugation, resuspended and incubated at
39.5 °C with 5 × 105 cells/ml in 50 ml flasks (Costar) and incubated for 48 h. One ml aliquots were removed ~/t
timed intervals and centrifuged at 400 g for 8 min, and the supernatants were saved for virus titration. Four
additional 200 ~tl aliquots were cytocentrifuged onto glass slides (Shandon Southern Instruments), fixed in
methanol :acetone (1:1) at - 2 0 °C for 5 min and stored at - 2 0 °C for immunoperoxidase staining.
Adherent cells cultured in Petri dishes were inoculated with 108.4 EID5o Ty/Ont and incubated for 45 min at
39-5 °C. The dishes were rinsed three times with medium and incubated at 39.5 °C with 5 ml fresh medium. All
media were removed at timed intervals for virus titration and replaced by fresh media.
Virus titration. To determine titres of infectious virus in the spleens of inoculated chickens, tissues were weighed
and then disrupted with a Stomacher (Tekmar Company). Suspensions and supernatants from inoculated cell
cultures were titrated in embryonated chicken eggs as previously described (Hinshaw et al., 1978). After 48 h at
35 °C, virus in allantoic fluid was detected by haemagglutination with chicken erythrocytes.
Infectious centre assay. To provide lymphocyte and adherent cell cultures infected in vivo, a chicken was
inoculated intratracheally with 9.5 x 103 p.f.u./0-5 ml BHIB, killed 24 h post-infection (p.i.) and the spleen was
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removed. Lymphocytes were cultured in suspension at 1 x 107 cells/ml. Adherent cells were prepared in six-well
tissue culture plates. Aliquots of lymphocytes or the adherent cells from one well were removed at timed intervals,
diluted and inoculated onto confluent monolayers of Madin-Darby canine kidney cells. After incubation at 37 °C
for 30 min, the cells were overlaid with agar containing trypsin (1 lag/ml), incubated for 48 h, and then overlaid
with agar containing neutral red. Plaques were counted after 24 h.
lmmunoperoxidase staining. Paraffin-embedded tissues were deparaffinized through a xylene and ethanol series.
Cytocentrifuged slides and two-chambered slides fixed in methanol:acetone (1:1) were stained directly. Viral
antigen was detected using the ABC avidin-biotin-immunoperoxidase staining technique (ABC Kit, Vector
Laboratories) and a monoclonal antibody (MAb) to the viral nucleoprotein (NP) prepared in this laboratory. T
lymphocytes were identified using a MAb to the avian T3 surface molecule kindly provided by Drs Chen-lo Chen
and Max D. Cooper (Chen et al., 1986). Cells bearing surface Ia molecules were detected using the MAb CIa-1
(Fisher Scientific).
Radioimmunoprecipitation (RIP). Virus-inoculated cultures of lymphocytes or adherent cells were incubated in
methionine-free medium with 10~ BSA supplemented with 0.1 mCi of [35S]methionine for 8 h at 39.5 °C. Cell
lysates were prepared and radioimmunoprecipitation was performed using MAbs to the NP and H5
haemagglutinin (HA) of Ty/Ont prepared in our laboratory. Precipitated antigens were separated using 10 ~oSDSPAGE (Laemmli, 1970) and examined by autoradiography.
In situ hybridization. The probe used in these procedures is a BamHl restriction enzyme fragment of the NP
clone, pSP56/501m, from A/PR/8/34 which was generously provided by Drs M. Krystal and P. Palese, Mount
Sinai School of Medicine, New York, U.S.A. An RNA probe labelled with [35S]UTP was prepared from this
fragment in a 50 lal volume containing 40 mM-Tris-HC1 pH 7.5, 6 mM-MgC12, 2 mM-spermidine, 10 mMdithiothreitol, 0-5 mM each of ATP, CTP and GTP, 2 lag DNA, 6 laM-UTP, 2'5 laM-[3sS]UTP and 10 units of SP6
RNA polymerase. In situ hybridization was performed on paraffin-embedded tissue sections as described by
Haase (1987) with the following modifications. Treatments to permeabilize tissue sections with digitonin, to
remove RNA with ribonucleases A and T1, and to post-fix dsDNA with paraformaldehyde were eliminated. The
hybridization solution contained 50~ deionized formamide, 0-6 M-NaCI, 10 mM-Tris-HC1 pH 7.4, l mM-EDTA,
50 lag/ml calf thymus DNA and 50 lag/ml yeast RNA in 1 x Denhardt's solution. The sections were pretreated
with hybridization solution for 2 to 4 h at 37 °C, then incubated with the probe in the same solution, supplemented
with 0.1 ~ Triton X-100 for 72 h. Slides were washed four times for 30 rain with 1 mM-EDTA and 0.1 ~ Triton X100 in 2 x SSC buffer, then washed once for 30 min in 50~ deionized formamide, 0.6 M-NaC1, 10 mM-Tris-HCl
pH 7.4, 1 mM-EDTA. Slides were then washed twice in SSC for 45 rain, followed by four washes for 30 rain in
0.6 M-NaCI, 10 mM-Tris-HC1 pH 7.4, 1 mM-EDTA. Sections were dehydrated in 70~ and 95 ~oethanol with 0.3 Mammonium acetate, air-dried, coated with emulsion (Ilford) and stored at 4 °C for 1 week. The slides were
developed for 8 min, rinsed, fixed for 15 rain, rinsed for 30 min and counterstained with haematoxylin.
Characterization of adherent cells. Adherent cells on two-chambered slides were fixed in citrate-acetonemethanol fixative and analysed for ct-naphthyl acetate esterase activity using a Histozyme kit 90-1A (Sigma).
Adherent ceils fixed in acetone-methanol were stained with Diff-Quik (American Scientific Products) and
examined for phagocytosis of erythrocytes by light microscopy. Immunoperoxidase staining using the MAb CIa-1
was performed as described above.
Proliferation assay. Spleen cell cultures were inoculated with 1, 10 and 100 EIDs0/cell of Ty/Ont, or with
uninfected allantoic fluid and incubated for 45 min at 39.5 °C. Five x l0 s cells in 100 lal of culture medium were
placed into wells of 96-well U-bottomed tissue cultures plates (Costar). Six wells were prepared with 100 p.1 of
medium or medium supplemented with 10 lag/ml of concanavalin A (Con A; Sigma) and then incubated for 43 h.
Wells were pulsed with 0.5 laCi of [3H]thymidine (New England Nuclear) in 5 lal of medium, cultured for an
additional 5 h and cells were harvested onto glass fibre filters using a PhD cell harvester (Cambridge Technology
Company). Incorporated radioactivity (c.p.m.) was counted in a scintillation medium (WestChem).
RESULTS
Effect of virulent avian influenza H5 viruses on the lymphoid tissues of chickens
T o d e t e r m i n e w h e t h e r the destructive effect of T y / O n t o n a v i a n l y m p h o i d tissues was
characteristic o f o t h e r v i r u l e n t strains o f influenza virus, we e x a m i n e d tissues o f c h i c k e n s
i n f e c t e d w i t h t w o o t h e r v i r u l e n t a v i a n H 5 viruses, T e r n / S . A . and C k / P e n n . In c o n t r a s t to the
e x t e n s i v e l y m p h o i d necrosis o b s e r v e d in c h i c k e n s i n o c u l a t e d w i t h T y / O n t (Van C a m p e n et al.,
1989), only three o f 10 c h i c k e n s i n o c u l a t e d w i t h T e r n / S . A . d e m o n s t r a t e d i n v o l v e m e n t o f the
spleen, limited to a few focal areas o f h e t e r o p h i l i c infiltrates a n d l y m p h o i d necrosis. F o u r birds
i n o c u l a t e d w i t h C k / P e n n did n o t d e v e l o p l y m p h o i d necrosis in any o r g a n e x a m i n e d . F i n d i n g s of
m y o c a r d i t i s and encephalitis in c h i c k e n s i n o c u l a t e d w i t h T e r n / S . A . a n d p a n c r e a t i t i s and
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(a)
H. VAN CAMPEN, B. C. EASTERDAY AND V. S. HINSHAW
(b)
Fig. 1. (a) Immunoperoxidasestaining of the spleen from a chicken infected with Ty/Ont. The brown
stain localized the viral nucleoprotein in discrete, round cells in a section of spleen. (/7)
Immunoperoxidase staining of a section of spleen from a chicken infected with Tern/S.A. The viral
antigen represented by the brown colour is detected in endothelial cells of an arteriole. Bar marker
represents 50 ktm.
myocarditis in chickens inoculated with Ck/Penn were consistent with previous reports (Acland
et al., 1984; Uys & Becker, 1967). Thus the destructive effect of Ty/Ont on lymphoid tissues is
characteristic of this particular influenza A virus, but not of other virulent H5 viruses.
To correlate the differences in the tissue damage caused by these H5 viruses with their
replication in different cell types, sections of spleen were examined for the presence of viral
antigen by immunoperoxidase staining. In spleens from Ty/Ont-infected chickens, viral antigen
was localized to discrete, round cells in areas of necrosis (Fig. 1 a). With Tern/S.A., viral antigen
was present in endothelial cells of vessels throughout the spleen, as well as in cells in areas of
necrosis (Fig. 1 b). In contrast viral antigen was rarely detected in the spleens of chickens
infected with Ck/Penn and, when seen, was restricted to the endothelial cells. Therefore virulent
avian influenza viruses infect and replicate in different cell types in vivo. The presence of viral
antigen in lymphoid cells is characteristic only of the highly virulent Ty/Ont and not of these
other virulent H5 viruses.
Infectious virus production by lymphocytes and adherent cells
The results of histopathological studies suggested that the destruction of lymphoid tissue in
Ty/Ont-infected chickens was due to productive, lytic infection of cells found in lymphoid tissue
(Van Campen et al., 1989). To determine the specific cells involved, avian spleen cells were
separated into lymphocytes and adherent cells. The latter were characterized further: cultures of
adherent cells from the spleens contained large cells with abundant cytoplasm and cytoplasmic
processes, which stained positively for non-specific esterase activity and phagocytized
erythrocytes (data not shown). Based on immunoperoxidase staining, these cells possessed the Ia
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2
3
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4
HAO
HAl
HA2
Fig. 2. Autoradiographof immunoprecipitates fromcultures of Ty/Ont-inoculatedadherent cells (lanes
1 and 2) and lymphocytes(lanes 3 and 4). At 1 h p.i., cells were labelled with [3sS]methionine for 6 h.
Cell lysates were reacted with MAbs to the nucleoprotein (NP) (lanes 1 and 3) or to the H5
haemagglutinin (HA) (lanes 2 and 4), and the immunoprecipitated proteins were resolved by SDSPAGE. The locationof the NP, HA0 and the subunits HA1 and HA2 are indicated on the left margin.
The Mr values of the electrophoretic markers are indicated alongthe right margin. The largest band in
the adherent cell lysatesprecipitated with MAb to NP and may represent an NP-polymerasecomplex.
antigen of macrophages, but not the T3 antigen of T lymphocytes. These adherent cells shared
many characteristics with macrophages and are, hereafter, designated as such.
To determine whether splenic lymphocytes or macrophages could support productive
infection with Ty/Ont, cultures of these cells were inoculated in vitro and assayed for infectious
virus. Titres of virus in the supernatants decreased over 48 h incubation, indicating that
infectious virus was not produced in either lymphocyte or macrophage cultures (results not
shown). In infectious centre assays, virus was detected in both macrophages and lymphocytes
from the inoculated chicken, indicating that these cells were infected in vivo. The number of
infectious centres decreased over 48 h in culture; thus, these cells did not produce infectious
virus in vitro (results not shown). We recognized that our culture conditions might be
inappropriate for productive infection, so we examined a variety of different factors for their
effect on virus production, including media, serum source, incubation temperature, cell
concentration and prior activation of lymphocytes with mitogen. The cell cultures still failed to
produce infectious virus.
In vitro production of viral proteins in lymphocytes compared to macrophages
To determine whether viral proteins were produced in vitro, the lymphocyte and macrophage
cultures were inoculated with Ty/Ont and assayed for viral antigen by immunoperoxidase
staining; lymphocytes, including T lymphocytes identified by immunoperoxidase staining for
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H. VAN CAMPEN, B. C. EASTERDAY AND V. S. HINSHAW
Fig. 3. Detection of viral RNA by in situ hybridization in a large cell within the white pulp of a spleen
from a chicken 60 h p.i. with Ty/Ont. A [35S]UTP-labelled RNA probe to the viral NP gene was used.
Bar marker represents 50 Ixm.
the T3 molecule, did not stain for viral antigen (results not shown). In contrast, viral antigen was
found in the cytoplasm of macrophages inoculated with Ty/Ont. The staining was not due to
phagocytosed virus since macrophages inoculated with inactivated virus failed to stain (results
not shown).
RIP of cell lysates with MAbs to the H5 HA and NP, shown in Fig. 2, indicated that infected
macrophages produced these viral proteins. Furthermore, the HA molecules produced in
macrophages were cleaved into HA1 and HA2 subunits (Fig. 2); such cleavage of the HA is a
characteristic of virulent avian influenza viruses and a prerequisite to their infectivity (Klenk et
al., 1975; Lazarowitz & Choppin, 1975). A small amount of viral NP, but no HA, was detected
in cell lysates of lymphocyte cultures, indicating either a low level of viral protein synthesis or
synthesis in a small subpopulation of these cells. We cannot exclude the possibility that the NP
may have been synthesized in a few contaminating macrophages. In the RIP of lymphocyte
cultures, additional bands were seen (Fig. 2) and their origin, that is, cellular or viral, is not
known; their precipitation appears to be non-specific as they are precipitated by both MAbs.
These results indicate that in vitro cultures of macrophages and lymphocytes synthesize viral
proteins, although no infectious virus particles are produced.
Production of viral RNA in lymphocytes and macrophages
To detect the presence of viral RNA, lymphocyte and macrophage cultures were inoculated
with Ty/Ont and examined by in situ hybridization. Viral RNA was detected in macrophages,
but not in lymphocytes (not shown). In sections of spleen from Ty/Ont-inoculated chickens, viral
RNA was also detected in a small number of large cells in the areas of lymphoid necrosis (Fig. 3).
These cells appeared to be macrophages on the basis of their size and distribution. Thus viral
RNA was evident in macrophages in vivo and in vitro.
Effect of Ty/Ont on the in vitro proliferation of Con A-stimulated lymphocytes
To determine whether Ty/Ont affects avian lymphocyte function, cultured spleen cells were
inoculated with different doses of Ty/Ont and then compared for their ability to proliferate in
response to the T lymphocyte mitogen, Con A. The proliferative response of lymphocytes as
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Pathogenesis o f virulent avian influenza
Table 1. Con A stimulation of chicken spleen cells inoculated with Ty/Ont or fl-propiolactoneinactivated virus
[3H]Thymidineincorporation (c.p.m.)
t"
Virus
Ty/Ont
Ty/Ont + Con A
Inactivated Ty/Ont
Inactivated Ty/Ont +
Con A
h
Uninfected
control
•
1
Dose of virus (EIDso/cell)
~"
10
294 + 124"
61398 __+25795
296 +_ 93
405 +_ 96
100247 + 11130
441 + 138
486 _+ 106
93756 _+ 14274
350 + 58
58287 4-_ 35083
120666 _+ 21979
116435 + 10468
100
174 _ 74
607 + i86
184 _ 75
152 + 68
* Average of six replicate wells + s,D.
measured by the incorporation of[3H]thymidine (Table 1) did not increase with increasing doses
of virus alone, indicating that Ty/Ont, itself, was not mitogenic. In the presence of Con A, doses
of 1 and 10 EIDs0 per cell of Ty/Ont resulted in marked enhancement of the response compared
to that of the uninfected controls, The enhancement appeared to be synergistic, being greater
than the sum of the responses to virus or to mitogen alone. A clearly different effect was found
with cells inoculated with the highest dose of Ty/Ont, i.e, 100 EIDso per cell resulted in severe
inhibition of the proliferative response of these cells to Con A compared to the uninfected
control. Addition of fl-propiolactone-inactivated virus to cell cultures gave similar results
indicating that replication was not necessary for enhancement or for inhibition of lymphocyte
proliferation in response to mitogen.
To determine whether spleen cells infected in vivo were similarly affected, chickens were
inoculated intratracheally with 103 EIDso of Ty/Ont, then killed at 12, 24 and 48 h later; the titre
of infectious virus per g of spleen was 0, 2-5 x 105 and 3-2 x 106 EID5o respectively. The spleen
cells from inoculated chickens were assayed for their ability to respond to Con A. The
proliferative responses of cells (mean + s,E,~, of six replicate cells) harvested 12 h (30712 +
3913 c.p.m.) and 24 h (95253 + 14786 c.p.m,) p.i. were markedly stimulated compared to the
uninfected control (8120_+ 1312 c.p.m.). In contrast, the response of cells 48 h p.i. (431 _+104
c.p.m.) was severely depressed, Thus, the proliferative response of avian lymphocytes
inoculated with Ty/Ont in vivo behaved in a manner similar to cells inoculated in vitro. Low doses
of Ty/Ont appeared to activate lymphocytes and to enhance their ability to respond to mitogen,
whereas high doses of Ty/Ont depressed the mitogenic response.
DISCUSSION
Destruction of lymphoid tissues by Ty/Ont is a characteristic of infection with this highly
virulent avian influenza virus and not of other virulent avian H5 viruses, Tern/S.A. or Ck/Penn.
These three strains vary in the cell type(s) in which viral antigen is found, indicating that they
infect and replicate in different cell types. The resulting differences in the type and degree of
tissue damage may explain the variable manifestations of disease associated with these viruses
(Easterday & Beard, 1984). Our findings indicate that the pathogenesis of virulent avian
influenza viruses is likely to involve more than one mechanism; therefore, conclusions drawn
from investigations of one strain may not be applicable to others.
Previous in vivo studies (Van Campen et al., 1989) suggested that the destruction of lymphoid
tissues in chickens infected with Ty/Ont was the result of lytic and productive infection of
lymphocytes. Our in vitro experiments indicate that splenic macrophages, as well as
lymphocytes, are infected by this virus and support the synthesis of viral proteins. Free
infectious virus is not produced by either cell type in vitro; the reason for this is not yet clear. In
situ hybridization detected viral RNA in macrophages infected with Ty/Ont both in vivo and in
vitro, emphasizing the importance of the macrophage in the viral infection.
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H. VAN CAMPEN, B. C. EASTERDAY AND V. S. HINSHAW
The striking feature of infection with Ty/Ont is the destruction of lymphoid tissues. This could
occur by virus infection that results in killing lymphocytes and macrophages present in large
numbers in the spleen; however, processes other than viral replication might be involved. In
investigating this possibility, we found that Ty/Ont affected the in vitro response of avian
lymphocytes to mitogen in a dose-dependent manner. Possible explanations for the enhanced
response with low doses of Ty/Ont include the release of lymphocyte-activating factors by
macrophages, direct activation o f lymphocytes or a direct mitogenic effect of influenza virus on
lymphocytes. W e favour the first possibility, i.e. that infected macrophages release soluble
factors that activate adjacent lymphocytes and enhance their ability to respond to mitogen. In
support of this, h u m a n macrophages produce intedeukin-I (Roberts et aL, 1986) in response to
influenza A virus; therefore avian macrophages may produce interleukin-1 and other cytokines
in response to Ty/Ont. Direct activation of lymphocytes by Ty/Ont cannot be excluded since
they a p p e a r to be infected by the virus; however, studies by Roberts (1982) suggest that effects of
influenza A viruses on lymphocyte proliferation are macrophage-dependent. It is unlikely that
Ty/Ont has a direct mitogenic effect as has been demonstrated for viruses of the H2, H3 and H6
subtypes for murine B lymphocytes (Poumbourios et al., 1987) because addition of Ty/Ont alone
did not increase the proliferation of the lymphocytes.
The highest dose of Ty/Ont resulted in a profound inhibition of the lymphocyte response to
mitogen. This inhibition of function m a y reflect a decrease in the viability of the lymphocytes, as
well as direct inhibition of lymphocyte function. The destruction of lymphocytes in vivo and
decreased viability of lymphocytes and macrophages observed in vitro suggest that Ty/Ont
affects the viability of these cells. Influenza A viruses are reported to be toxic as a result of
abortive infections of non-permissive cells (Mims, 1960; Rodgers & Mims, 1981). The observed
inhibition with Ty/Ont cannot be fully explained by abortive infection of the lymphocytes, as a
decrease response to Con A also occurred with inactivated virus. W e speculate that Ty/Ont
infection of macrophages m a y well affect the viability of lymphocytes. Macrophages exposed to
influenza virus release toxic substances, such as oxygen radicals (Oda et al., 1989), lysosomal
contents or soluble factors, such as tumour necrosis factor (Beutler et al., 1986). Therefore the
lymphoid necrosis observed in vivo m a y be due to the release of deleterious factors by infected
macrophages, as well as to viral replication. F u r t h e r investigation of the interaction between
influenza viruses and target cells, such as lymphocytes and macrophages, may help us
understand the effects of other acute viral infections on lymphoid tissues.
The authors thank Drs Chen-lo Chen and Max D. Cooper, Tumor Institute, University of Alabama for
providing the monoclonal antibody to the avian T3 molecule. We appreciate the technical assistance of M. S.
Sheerar, T. Ruffalo and N. Dybdahl-Sissoko and the helpful discussions with A. Haase, A. Palmenberg, M. S.
Philpott and C. Hioe. This work was supported by the United States Department of Agriculture Biotechnology
Grant 86-CRCR- 1-2194, Special Grant CRSR-2-3159 and Wisconsin Agricultural Experiment Station WIS3101,
and was presented in part of the American Association of Avian Pathology Meeting in July 1988.
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