Journal of General Virology (2007), 88, 1275–1280 DOI 10.1099/vir.0.

82423-0

Short Differential onset of apoptosis in influenza A virus

Communication H5N1- and H1N1-infected human blood
macrophages
Chris K. P. Mok,1 Davy C. W. Lee,1 Chung-Yan Cheung,2 Malik Peiris2
and Allan S. Y. Lau1
Correspondence 1
Immunology Research Laboratory, Department of Paediatrics and Adolescent Medicine,
Allan S. Y. Lau Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR,
asylau@hkucc.hku.hk China
2
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Pok Fu Lam, Hong Kong SAR, China

Received 28 July 2006
Accepted 14 December 2006
Pathogenesis of the highly pathogenic avian influenza virus A/Hong Kong/483/97 (H5N1/97)
remains to be investigated. It was demonstrated recently that H5N1 dysregulation of
proinflammatory cytokines in human macrophages is a p38-kinase-dependent process. The
results indicated that macrophages may play a role in disease severity. To investigate cellular
responses to H5N1 infection further, apoptosis and its related pathways were studied in primary
blood macrophages. Here, it is shown that the H5N1/97 virus triggered apoptosis, including
caspases and PARP activation, in infected macrophages with a delayed onset compared with
H1N1 counterparts. Similar results were also found in human macrophages infected by
precursors of the H5N1/97 virus. Thus, these results showed that the delay in apoptosis onset
in macrophages infected by H5N1/97 and its related precursor subtypes may be a means for
the pathogens to have longer survival in the cells; this may contribute to the pathogenesis of
H5N1 disease in humans.

The H5N1/97 ‘bird-flu’ incident was the first documented
direct transmission of an avian influenza virus to humans,
causing devastating infections with severe viral pneumonia
and a mortality rate of .30 % (Claas et al., 1998; Subbarao
et al., 1998; Yuen et al., 1998). The H5N1 virus continued
to disseminate among migratory birds and led to a wide-
spread outbreak in domestic poultry around the world.
It had caused over 200 human cases as of May 2006
(WHO avian influenza information; http://www.who.int/
csr/disease/avian_influenza/en/). The increasing incidence
of avian-to-human transmission provides an opportunity
for these highly pathogenic avian influenza viruses to adapt
to the host environment, which may result in reassortment
of genetic materials between avian and human influenza
viruses. Generation of such newly reassorted influenza
virus may lead to a potential pandemic threat (WHO avian
influenza information; http://www.who.int/csr/disease/
avian_influenza/en/).
Previously, we demonstrated that H5N1/97 viruses, in
contrast to human influenza A virus subtypes including
H1N1 and H3N2, induce high levels of proinflammatory
cytokines in differentiated primary human blood macro-
phages (Cheung et al., 2002). It was suggested that this
Supplementary figures are available in JGV Online.
cytokine dysregulation contributes to pathogenesis and
severity of the disease (Fisman, 2000; Headley et al., 1997;
To et al., 2001). In delineating the mechanisms of cytokine
dysregulation, we recently reported that p38K, a mitogen-
activated protein kinase (MAPK), plays a significant role
in the hyperinduction of tumour necrosis factor alpha
(TNF-a) in H5N1-infected macrophages (Lee et al., 2005).
In addition, a recent study showed that H5N1-infected
macrophages enhance TNF-related apoptosis-inducing
ligand (TRAIL)-induced apoptosis in Jurkat T cells (Zhou
et al., 2006). These recent reports indicate that human
blood macrophages may play a critical role in the patho-
genesis of H5N1 infection.
Among cellular responses against invading viruses, induc-
tion of apoptosis has been postulated to be one of the most
effective host defence mechanisms. The cell-death process
results in inhibition of virus replication, limitation of virus
dissemination and minimization of uncontrolled inflam-
matory responses. It has been shown that influenza virus
induces apoptosis in vivo and in vitro (Brydon et al., 2005;
Fesq et al., 1994; Hinshaw et al., 1994; Takizawa et al., 1993,
1999). However, the functional role of influenza virus-
induced apoptosis is still not well defined. To gain insights
into the virulence of the highly pathogenic avian influenza
virus, together with an understanding of the cellular

0008-2423 G 2007 SGM Printed in Great Britain 1275

C. K. P. Mok and others
responses of macrophages during microbial infection, we
studied the mechanisms of apoptosis in primary blood
macrophages infected with H5N1 (483/97) or human
influenza virus H1N1 (54/98).
Primary blood macrophages were isolated from mono-
nuclear cells of healthy blood donors as described pre-
viously (Lee et al., 2005). The cells were mock-treated or
infected with influenza viruses (H5N1 or H1N1) at an
m.o.i. of 2 for 30 min and harvested at indicated time
points for analysis. The infectivity of H5N1 and H1N1
viruses on human macrophages was examined by immuno-
fluorescent staining using monoclonal antibodies specific
for the nucleoprotein of influenza viruses (DAKO).
Positive staining for the nucleoprotein in influenza virus-
infected macrophages was found at 8 h post-infection
(data not shown). Additionally, cell death, as demonstrated
by nuclear condensation or nuclear fragmentation, was
determined by staining the cells with 4,6-diamidine-2-
phenylindole dihydrochloride (DAPI) at indicated time
points post-infection. As shown, the number of dead cells
was lower in those cells infected by the H5N1 virus than in
cells infected by its H1N1 counterpart (Fig. 1a).
A previous report showed that replication of influenza
virus is necessary to trigger apoptosis in infected cells
(Price et al., 1997). We thus measured the viral titres of
H5N1 or H1N1 in the infected macrophages at 6, 10, 18
and 24 h post-infection. There was no significant difference
between the viral titres in the macrophages infected by
H1N1, H5N1 (Fig. 1b) or A/Quail/Hong Kong/G1/97
(H9N2/G1), the precursor of H5N1/97 (see Supplementary
Fig. S1, available in JGV Online). Hence, the delayed onset
of apoptosis in H5N1-infected macrophages was not due to
replication of the avian virus. Furthermore, we performed
kinetic studies to measure the level of the viral genes for the
polymerase protein (PA) and nucleoprotein (NP) in
H5N1- or H1N1-infected macrophages by using a quanti-
tative RT-PCR assay. Consistent with the virus titration
results, we did not find any significant differences in the
transcription levels of PA and NP in H5N1- or H1N1-
infected macrophages at 0, 6 or 10 h post-infection (see
Supplementary Fig. S2, available in JGV Online).
We next examined the ultrastructural features of H5N1-
and H1N1-infected cells at 12 h post-infection under
a transmission electron microscope (Philips EM208S).
The cellular morphology of H5N1-infected macrophages
(Fig. 1c, middle) was comparable to that of mock-treated
cells at 12 h post-infection (Fig. 1c, right). In contrast,
H1N1-infected cells showed characteristics of apoptotic
cells, including nuclear condensation and chromatin
adherence to the nuclear membrane (Fig. 1c, left). Our
results demonstrated a differential onset of cell death in
macrophages infected with H5N1 compared with those
infected with the H1N1 virus.
We investigated the apoptotic pathways in virus-infected
macrophages further by measuring the activation of
caspase-activated poly(ADP–ribose) polymerase (PARP;
1276
Pharmingen), an essential factor in the initiation of
apoptosis (Soldani & Scovassi, 2002), at 6, 12 and 18 h
post-infection by using Western analysis (Fig. 1d). Over the
time course, there was a 4- or 8-fold increase in the levels of
the cleaved PARP fragment in H5N1-infected cells at 12 or
18 h post-infection, respectively, compared with the mock-
treated cells (Fig. 1d, lanes 6 and 7). In contrast, there
was an 18- or 22-fold increase in PARP fragment levels
in H1N1-infected cells at the corresponding time point
(Fig. 1d, lanes 3 and 4). As PARP is the downstream target
of the caspase cascade, we then measured the activity of
two important apoptotic markers, caspases 3 and 8, by
Western analysis using antibodies from Cell Signaling
Technology and Upstate Biotech, respectively. Our results
showed that the cleaved fragment of caspase 3 was barely
detectable in cells infected with H5N1 (Fig. 2a, lane 9) and
was not detected in those infected with H9N2/G1 (data
not shown) at 12 h post-infection. In contrast, levels of
activated caspase 3 were increased significantly in H1N1-
infected cells at 10 h post-infection and persisted at 12 h
(Fig. 2a, lanes 4–5). Consistent with the caspase 3 findings,
H5N1 did not induce the degradation of procaspase 8
strongly at 12 h post-infection compared with H1N1
(Fig. 2b, lanes 9 and 5).
We further examined the functional role of caspase 8 in
influenza virus-induced apoptosis by treating virus-infected
cells with a specific inhibitor for caspase 8, Z-IETD-FMK
(Takizawa et al., 1999). Following caspase 8 inhibition, our
results showed that the level of the cleaved PARP fragment in
H5N1- or H1N1-infected macrophages was reduced sig-
nificantly, by 82 and 86 %, respectively, compared with the
corresponding samples without inhibitor treatment (Fig. 2c,
lanes 3 and 5). Therefore, the differential onset of cell death
in H5N1 and H1N1 infection was associated with the delayed
activation of the caspase cascade.
To investigate the characteristics of the onset of apoptosis
in avian influenza virus infection further, we measured the
levels of PARP activation in macrophages infected with
precursors of H5N1/97, including H9N2/G1, A/Teal/HK/
W312/97 (H6N1) and A/Goose/Guangdong/1/96 (H5N1/
437.6) (Guan et al., 1999; Hoffmann et al., 2000; Subbarao
& Shaw, 2000; Xu et al., 1999), and human influenza viruses,
including H1N1 and H3N2, at 18 h post-infection (Fig. 3a,
b). The level of activated PARP in macrophages infected with
the H5N1/97 virus or its precursors was lower than that in
H1N1- or H3N2-infected cells. Our findings suggested that
macrophages infected with H5N1/97 or its precursors
undergo slower kinetics of apoptotic responses compared
with H1N1- or H3N2-infected cells. In addition, H9N2/G1
induced the cleavage of PARP, but not that of caspase 3,
suggesting that H9N2/G1 activates PARP through a caspase
3-indpendent pathway (Hong et al., 2004).
The detailed mechanisms of delayed onset of apoptosis
occurring in avian influenza virus-infected human macro-
phages remain to be investigated. It is plausible that the
infected macrophages cannot recognize the avian influenza
Journal of General Virology 88
 Delayed onset of apoptosis in H5N1-infected macrophages

(a) 60 (b) 6

Virus titre [log10 (TCID50 ml 1)]

H1N1

_
50 5 H5N1

Cell death (%)

40 4

30 3

20 2

10 1

H1N1 H5N1 Mock 6 10 18 24

Time (h)

(c)

CC

H1N1 H5N1 Mock

(d) Mock H1N1 H5N1

_
Time post-infection (h) 6 12 18 6 12 18
Pro-PARP
Cleaved PARP

Fold induction (1.00) (1.30) (17.41) (21.71) (0.15) (4.36) (8.37)

Actin
(1) (1) (1) (1) (1) (1) (1)

1 2 3 4 5 6 7

Fig. 1. H5N1/97-infected macrophages show less cell death and apoptosis than H1N1-infected macrophages. (a) Primary
human blood macrophages (0.5¾106) were fixed with 4 % paraformaldehyde and stained with DAPI at 18 h post-infection with
H5N1 (483/97) or H1N1 (54/98) virus at an m.o.i. of 2. Values represent the mean±SD of cells from three different donors
analysed statistically by the two-tailed, paired t-test. *P , 0.1. (b) Replication of the H5N1 (483/97) virus in primary human
blood macrophages. Primary human blood macrophages (1¾106) were infected with human influenza virus H1N1 (54/98) or
avian influenza virus H5N1 (483/97) at an m.o.i. of 2. Culture supernatants were collected at 6, 10, 18 and 24 h post-infection.
Viral titres (TCID50) of the samples were measured by titration in Madin–Darby canine kidney (MDCK) cells. The results shown
are representative of experiments performed on cells from three different donors. (c) Primary human blood macrophages
(2¾106) were infected with influenza virus H5N1 (483/97) or H1N1 (54/98) at an m.o.i. of 2. At 12 h post-infection, the cells
were collected for ultrastructural examination by transmission electron microscopy. H1N1-infected macrophages (left); H5N1-
infected macrophages (middle); mock-treated macrophages (right). Magnification, ¾28 000; CC, chromatin condensation. The
results shown are representative of experiments performed on cells from three different donors. (d) Primary human blood
macrophages (1¾106) were harvested at 6, 12 and 18 h after infection with H5N1 (483/97) or H1N1 (54/98) virus at an m.o.i.
of 2. The activated level of PARP was assayed by Western analysis using a monoclonal anti-PARP antibody. Equal loading of
protein samples was demonstrated with anti-actin antibodies. The results shown are representative of experiments performed
on cells from three different donors. The density of the protein band was determined by using Bio-Rad Quantity One imaging
software. Numbers in parentheses are density values of activated PARP relative to that of actin. Mock, uninfected cells.

virus effectively and trigger apoptosis efficiently to fight H3N2 viruses (Cooper & Subbarao, 2000). Whether the
against the invading avian influenza virus. It has been variation of the viral genome sequences affects the
documented that the sequences of H5N1/97 virus are quite interactions between the host and the virus remains to be
different from those of the human influenza H1N1 or determined.

http://vir.sgmjournals.org 1277
C. K. P. Mok and others

Fig. 2. (a) Delayed activation of caspase 3 in

H5N1-infected human macrophages. Primary
human blood macrophages (1¾106) were
(a) Mock H1N1 H5N1
harvested at 6, 8, 10 and 12 h post-infection
Time post-infection (h) _ 6 8 10 12 6 8 10 12 with H5N1 (483/97) or H1N1 (54/98) virus at
Procaspase 3 an m.o.i. of 2. Activation of caspase 3 was
assayed by Western analysis using polyclonal
anti-caspase 3 antibodies. (b) Activation of
Cleaved caspase 3 caspase 8 in H5N1-infected primary human
Fold induction (1.00)(1.87)(3.10)(13.50)(38.08)(1.53)(2.47)(4.51)(6.60) blood macrophages. Primary human macro-
phages (1¾106) were harvested at 6, 8, 10
Actin and 12 h post-infection with H5N1 (483/97)
(1) (1) (1) (1) (1) (1) (1) (1) (1) or H1N1 (54/98) virus at an m.o.i. of 2. The
1 2 3 4 5 6 7 8 9 degradation level of procaspase 8 was
assayed by Western analysis using a mono-
(b) Mock H1N1 H5N1 clonal anti-procaspase 8 antibody. (c)
Involvement of caspase 8 in the apoptosis of
Time post-infection (h) _ 6 8 10 12 6 8 10 12 H5N1-infected human macrophages. Primary
Procaspase 8 human macrophages (1¾106) were pretreated
Fold induction (1.00)(0.99)(1.02)(0.75) (0.25)(0.99) (0.97) (0.73) (0.70)
or not with 50 mM caspase 8 inhibitor Z-IETD-
Actin FMK for 1 h at 37 6C and infected with H5N1
(483/97) or H1N1 (54/98) virus at an m.o.i. of
(1) (1) (1) (1) (1) (1) (1) (1) (1)
2, or left uninfected. Total proteins were
1 2 3 4 5 6 7 8 9
harvested at 18 h post-infection and assayed
(c) Mock Mock H1N1 H1N1 H5N1 H5N1 by Western analysis using anti-PARP anti-
_ _ _ body. Equal loading of protein samples was
Inhibitor + + + Time post-infection = 18 h
demonstrated by using anti-actin antibodies.
Pro-PARP
Cleaved PARP The results shown are representative of
Fold induction (1.17) (1.00) (2.34)(16.61)(1.30) (7.46) experiments performed on cells from three
different donors. The density of the protein
Actin band was determined by using Bio-Rad
(1) (1) (1) (1) (1) (1) Quantity One imaging software. Numbers in
1 2 3 4 5 6 parentheses are density values of activated
caspase 3, procaspase 8 or activated PARP
relative to that of actin. Mock, uninfected cells.

Induction of anti-apoptotic genes and pathways in H5N1-
infected macrophages is another possible mechanism
leading to the delayed onset of apoptosis. In our previous
study, we observed that p38 MAPK is activated differen-
tially in primary human macrophages after infection by
H5N1/97 virus. This activation was shown to be associated
with superinduction of TNF-a by H5N1 (Lee et al., 2005).
Moreover, it has been shown that double-stranded RNA
activation of p38 MAPK results in inhibition of the activity
of caspases 3, 8 and 9 (Tadlock et al., 2003). This may be
analogous to the situation in H5N1 infection of macro-
phages, in which preferential activation of p38 MAPK may
contribute to delayed apoptosis compared with H1N1
infection. However, apoptosis in H5N1-infected macro-
phages was not decreased by using the p38 MAPK-specific
inhibitor SB203580 (see Supplementary Fig. S3, available
in JGV Online). Recent findings showed that the interac-
tion of Ccl5 and Ccr5 induces anti-apoptotic signals for
macrophages during viral infection (Tyner et al., 2005). As
Ccl5 was highly induced in H5N1-infected human macro-
phages compared with those infected by human influenza
viruses (Cheung et al., 2002), further investigation is required
to examine whether this signalling cascade plays a role in the
delayed onset of apoptosis in H5N1-infected cells.
Our results also provide additional information on the
role of macrophages in the pathogenesis of H5N1 infec-
tion. H5N1-infected patients present with severe viral
pneumonia and lymphopenia. A recent study reported that
H5N1-infected macrophages could enhance TRAIL-induced
apoptosis in T cells compared with H1N1 infection (Zhou
et al., 2006). Therefore, our results suggest that the
delayed onset of apoptosis in H5N1-infected macrophages
may prolong the interaction of TRAIL-expressing macro-
phages with T cells to enhance the induction of apoptotic
cell death in the T cells.
In conclusion, we have provided experimental evidence
that avian influenza viruses, including H5N1/97 and its
precursors, trigger an apoptotic response mediated by
caspase activation that is similar to, but delayed compared
with, that induced by human influenza viruses including
H1N1 and H3N2. Our findings on the delay of apoptosis
in H5N1 viruses combined with superinduction of
proinflammatory cytokines may contribute, in part, to

1278 Journal of General Virology 88

 Delayed onset of apoptosis in H5N1-infected macrophages

Fig. 3. Differential time of onset of apoptosis

(a) in primary human blood macrophages infected
Time post-infection = 18 h H1N1 H3N2 H6N1 H9N2 by different avian influenza viruses. (a) Primary
/G1 blood macrophages (1¾106) were infected
Pro-PARP with H1N1 (54/98), H3N2 (1174/99), H6N1
Cleaved PARP (W312/97) or H9N2 (G1/97) virus at an m.o.i.
Fold induction (30.48) (25.29) (5.92) (12.02) of 2 or treated with mock reagents. Protein
extracts were collected at 18 h post-infection
Actin
and assayed by Western analysis using a
(1) (1) (1) (1) monoclonal anti-PARP antibody. (b) Protein
1 2 3 4 extracts of H5N1 (483/97), H5N1 (437.6/99)
(b) and H1N1 (54/98)-infected primary human
Time post-infection = 18 h H5N1 H5N1 H1N1 Mock blood macrophages, as well as mock-treated
(437.6/99) cells, were collected and assayed under the
Pro-PARP same conditions as in (a). Equal loading of pro-
Cleaved PARP tein samples was demonstrated with anti-actin
Fold induction (7.46) (5.72) (33.08) (1.00) antibodies. The results shown are representative
of experiments performed on cells from three
Actin different donors. The density of the protein band
(1) (1) (1) (1) was determined by using Bio-Rad Quantity One
1 2 3 4 imaging software. Numbers in parentheses are
density values of activated PARP relative to that
of actin. Mock, uninfected cells.

understanding how these novel viruses cause fatal disease
in humans.
Acknowledgements
This work was supported by research grants to A. S. Y. L. (HKU 7430/
03 M) and to M. P. (HKU 7459/03) from the Research Grants Council
of Hong Kong and the Research Fund for Control of Infectious
Disease to A. S. Y. L. (project no. 05050112), as well as the 2003 Vice
Chancellor’s Development Fund from The University of Hong Kong
to M. P.
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Journal of General Virology 88