The Multibasic Cleavage Site of the Hemagglutinin of Highly
Pathogenic A/Vietnam/1203/2004 (H5N1) Avian Influenza Virus Acts
as a Virulence Factor in a Host-Specific Manner in Mammals
Amorsolo L. Suguitan, Jr.,a* Yumiko Matsuoka,aYuk-Fai Lau,a* Celia P. Santos,aLeatrice Vogel,aLily I. Cheng,b* Marlene Orandle,b
and Kanta Subbaraoa
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA,aand Comparative Medicine
Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USAb
mice, ferrets, and African green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with
many parts of the world highlights the threat that these viruses
pose to public health from their potential to cause another pan-
demic. More than 500 laboratory-confirmed HPAI H5N1 infec-
tions have been recorded since 2003 in 15 countries, with a case-
fatality rate of nearly 60% (54). Most of these infections can be
traced to direct exposure to diseased poultry; human-to-human
transmission of the H5N1 virus remains limited to small family
clusters (20, 50). To date, the gene compositions of the H5N1
viruses isolated from infected humans have remained wholly
avian in origin and are poorly transmitted among individuals, in
contrast to the efficient transmission displayed by the swine-
origin influenza A H1N1 pandemic virus that has infected mil-
lions worldwide. As the HPAI H5N1 viruses are now endemic in
may become easily transmissible among individuals through ge-
netic reassortment with a human influenza virus or through ad-
aptation in an intermediate host. Understanding the basis of the
virulence of HPAI H5N1 viruses in humans therefore remains a
Although several virus genes and gene products contribute to
ability of the hemagglutinin (HA) protein and the distribution of
HA-activating proteases in the host are recognized as major viru-
lence factors (6, 17, 40). The HA0 precursor protein undergoes
posttranslational cleavage into two subunits, HA1 and HA2, to
facilitate the fusion of the viral and cellular membranes (25, 27,
53). The amino acid sequence upstream of the HA cleavage site as
is believed to regulate HA cleavability (23, 24). Most of the low-
at the HA cleavage site. These HAs are cleaved by extracellular
(HPAI) H5N1 viruses from domestic poultry to humans in
trypsin-like serine proteases secreted by nonciliated cells of the
respiratory epithelium in humans and gastrointestinal tract of
birds, resulting in local infections (32). Some HPAI viruses of the
H5 and H7 subtypes, on the other hand, contain multiple basic
amino acids around the cleavage site (MBS) of the HA molecule
(5) that can act as a recognition motif for subtilisin-like proteases
that recognize polybasic motifs (39). The ubiquitous distribution
utes to the propensity of HPAI viruses with the MBS motif to
disseminate and replicate in extrapulmonary organs of birds,
thereby expanding the range of tissues that they can infect. While
the role of MBS as a virulence motif in poultry is well established,
the influence of MBS on the virulence of HPAI viruses in mam-
mals remains unknown and is the focus of this study.
the HA, tissue tropism also plays a major role in the pathogenesis
and host-range restriction displayed by influenza A viruses. Be-
cause human and avian influenza viruses differ in their binding
preference to terminal sialic acids (SA) on host cells, the type and
distribution of these residues on cells of the respiratory and gas-
trointestinal tracts are associated with the susceptibility of differ-
Received 28 June 2011 Accepted 9 December 2011
Published ahead of print 28 December 2011
Address correspondence to Kanta Subbarao, firstname.lastname@example.org.
*Present address: Amorsolo L. Suguitan, Jr., MedImmune, Mountain View,
California, USA; Yuk-Fai Lau, Medical Countermeasures (Biological) Laboratory,
DMERI, DSO National Laboratories, Singapore, Republic of Singapore; Lily I. Cheng,
MedImmune, Gaithersburg, Maryland, USA.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virologyp. 2706–2714
ent hosts to influenza A virus infection (45). van Riel and col-
leagues (2007) reported that human influenza viruses attach to
alveoli while H5 avian influenza viruses preferentially target cells
in the bronchioles, type II pneumocytes, and alveolar macro-
binding preference is still insufficient to predict the severity of
disease outcome induced by H5N1 viruses, as both low-patho-
genicity and HPAI H5N1 viruses target the same types of cells
(51). Intriguingly, H5N1 viruses could infect and productively
replicate in human nasopharyngeal and oropharyngeal tissues ex
vivo despite the absence of detectable ?2,3-SA receptors on
these cells (30). These findings suggest that alternative, as-yet-
human upper respiratory tract and that H5N1 disease severity is
not totally dependent on the predicted tropism of the virus for
tissues of the lower respiratory tract (30).
Data from clinical observations and limited autopsies con-
ducted on fatal cases of human H5N1 infection suggest that ex-
tensive virus replication, systemic spread, and the induction of an
intense inflammatory response may all contribute to the patho-
genesis of H5N1 influenza (11). The intense inflammatory re-
sponse has been implicated in the development of acute respira-
tory distress syndrome (3) which in turn is the leading cause of
death among patients infected with the H5N1 virus. Gaining a
better understanding of the virulence determinants of HPAI
H5N1 viruses in mammalian models would provide significant
insights on developing effective intervention and therapeutic
strategies that could improve the clinical management of the dis-
the H5 HA of the HPAI A/Vietnam/1203/2004 (H5N1) virus on
its virulence in BALB/c mice, ferrets, and African green monkeys
(AGMs; Chlorocebus aethiops).
MATERIALS AND METHODS
Recombinant viruses. The removal of the MBS from the H5 HA gene of
and ?H5N1 viruses by reverse genetics, was previously described (43).
Briefly, a coculture of Madin-Darby canine kidney (MDCK) cells and
293T cells was transfected with the plasmids that carry the eight gene
segments of the desired influenza virus by lipofection. Virus stocks were
all gene segments were verified to be identical to the plasmids used in the
conducted using enhanced containment procedures in biosafety level 3
(BSL3) facilities approved for use by the U.S. Department of Agriculture
and the Centers for Disease Control and Prevention. All animal studies
were approved by the National Institutes of Health Animal Care and Use
Virus replication in vitro. Confluent monolayer cultures of MDCK
cells in 6-well plates were infected with either the H5N1 or the ?H5N1
virus at a multiplicity of infection of 0.001 in triplicate. Culture superna-
tants were collected at 12-h intervals up to 72 h and stored frozen at
?80°C prior to virus titration on MDCK cells.
Virus replication and pathogenicity studies in mice. To determine
the ability of the viruses to replicate in different organs of mice, groups of
6- to 8-week-old female BALB/c mice (Taconic, Germantown, NY) were
lightly anesthetized and infected intranasally (i.n.) with 125 50% tissue
culture infectious doses (TCID50) of either the H5N1 (equivalent to 50?
50% mouse lethal dose [MLD50]) or ?H5N1 virus in 50 ?l. Four mice
as previously described (44).
by suspension array technology using the Bio-Plex Pro mouse cytokine
23-plex assay kit system (Bio-Rad, Hercules, CA) according to the man-
To assess the impact of the H5 HA MBS on the population of circu-
of animals served as mock-infected controls and received 50 ?l of L15
medium. Whole-blood samples were collected daily from the tail of mice
until day 5 p.i. Differential WBC counts were determined by preparing
thin blood smears, staining the slides with the Hema-3 system stain
(Fisher Scientific), and calculating the percentage of each WBC type per
100 WBCs counted under an oil-immersion objective.
To evaluate the pathology induced by the H5N1 viruses in the lungs,
sections were prepared from formalin-fixed, paraffin-embedded tissues
and then stained with hematoxylin and eosin. The sections were analyzed
for pathological changes by a pathologist who was unaware of the inocu-
lum administered to each mouse.
(Triple F Farms, Pennsylvania) were infected i.n. with 107TCID50of the
biological (b) or reverse-genetics-derived (r) H5N1 or ?H5N1 virus.
Three animals from each group were euthanized on days 3 and 6 p.i., and
virus titers were determined in the nasal turbinates, lungs, and brain as
previously described. A section of the right lung was examined for histo-
pathologic changes. The remaining 3 ferrets in each group were followed
for clinical observations, including weight and temperature monitoring,
for 14 days (44).
Measurement of ferret cytokine mRNAs by real-time PCR. Whole-
blood samples from ferrets infected with bH5N1, rH5N1, and ?H5N1
MD) before infection (day 0) and on day 3 p.i. The blood samples were
processed, and total RNA was isolated according to the manufacturer’s
instructions. Total RNA from nasal washes from the same ferrets was
RNA quantities for each sample were determined using a Nanodrop
2000C spectrophotometer (Thermo Scientific), and the concentrations
were normalized by dilution in nuclease-free water. RNA samples were
reverse transcribed using a QuantiTect reverse transcription kit (Qiagen)
each cDNA sample was preamplified with PCR primers corresponding to
alpha interferon (IFN-?), MX-1, interleukin-2 (IL-2), IFN-?, IL-6, IL-4,
ISG-15, IL-5, IL-12, RIG-1, CXCL10, IL-1?, RANTES, and the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping
gene in a 10-?l reaction volume using a TaqMan PreAmp Master Mix kit
(Applied Biosystems) according to the manufacturer’s protocol. Follow-
(TE) buffer, pH 8.0.
containing 18 ?M primers and 5 ?M probe (final concentration of 900
nM [each] primer and 250 nM [each] probe). Fluidigm BioMark 48-
by-48 arrays were prepared according to the manufacturer’s instructions.
Briefly, 20? assay mixes were diluted 1:1 (vol/vol) with DA assay loading
reagent (Fluidigm, South San Francisco, CA) and 5 ?l was added to du-
H5 HA Cleavability and Pathogenicity in Mammals
March 2012 Volume 86 Number 5jvi.asm.org 2707
plicate assay inlets of the array. Five microliters of each sample reaction
mix was prepared by mixing 2? TaqMan Universal Mastermix (Applied
Biosystems), DA sample loading reagent, and 2.25 ?l of sample cDNA.
The sample mixes were loaded in duplicate per the manufacturer’s rec-
sample. After loading, PCR was performed under the following reaction
conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of
old setting of 0.65 using the Fluidigm real-time PCR analysis software.
The mean for the four replicate threshold cycle (CT) values of each ferret
gene was determined and normalized relative to the mean CTvalue of the
GAPDH housekeeping gene. Fold changes were calculated over baseline
CTvalues represented by preinfection samples.
used in the study which was conducted at Bioqual, Inc. (Rockville, MD).
Two groups of 6 monkeys each were anesthetized and infected intratra-
cheally and intranasally with 2 ? 106TCID50of either the H5N1 or
?H5N1 virus while one monkey was mock infected with L15 medium.
Nasal and pharyngeal swabs, tracheal lavage samples, whole blood, and
serum were collected from each monkey for virus titration, hematology,
and cytokine analysis, respectively. The body weight and temperature of
the monkeys were noted daily, and each animal was observed for clinical
symptoms. The mock-infected animal was sacrificed together with two
monkeys from each group on day 2 p.i., and from then on, 2 monkeys
from each virus-infected group were sacrificed on days 4 and 7 p.i. Nasal
turbinates, lungs, trachea, brain, liver, and spleen were sampled at nec-
ropsy for virus titration and pathological evaluation. Cytokine and
chemokine measurements in AGM serum were conducted using the Bio-
Plex Pro human cytokine 27-plex assay kit system (Bio-Rad, Hercules,
CA) according to the manufacturer’s instructions.
the H5N1 virus in vitro, cultures of MDCK cells supplemented
virus that lacks the MBS in its HA cleavage site (?H5N1) at a
multiplicity of infection of 0.001. Virus titers were determined in
culture supernatants collected at 12-hour intervals. The H5N1
virus consistently replicated to slightly higher titers than did the
the H5N1 virus in vitro.
was previously determined to be a virulence motif in mice—re-
moval of the H5 HA MBS was sufficient to render the highly
pathogenic H5N1 virus nonlethal to mice (15, 43). To evaluate
whether the H5 HA MBS confers any replication advantage in
vivo, groups of mice were infected with either the H5N1 or the
tory and extrapulmonary organs at different time points postin-
fection. The ?H5N1 virus replicated to about 100- to 1,000-fold-
lower titers in the nasal turbinates and lungs of mice than did the
H5N1 virus (Fig. 1A and B, respectively), suggesting that removal
of the H5 HA MBS not only alleviates the lethality of the H5N1
virus (43) but is also associated with a significant reduction in the
level of virus replication in the respiratory tract of mice. The H5
HA MBS was also associated with extrapulmonary virus dissemi-
nation in mice, as only the H5N1 virus replicated consistently in
the brain (Fig. 1C). These results are consistent with our previous
a major virulence determinant in mice (43).
The H5 HA MBS and the cytokine response in the mouse
lung. Elevated levels of several proinflammatory cytokines and
chemokines have been reported in mice following primary infec-
tion with HPAI H5N1 viruses (47, 49). As cytokine dysregulation
has been suggested as one of the contributing factors for the ex-
treme virulence of HPAI H5N1 viruses (11, 49), the cytokine and
chemokine profiles elicited by the H5N1 and ?H5N1 viruses in
the lungs of mice infected with 125 TCID50of either virus were
evaluated using a multiplex cytokine bead array assay. The con-
centrations of the pleiotropic cytokine granulocyte colony-
stimulating factor (G-CSF), as well as the proinflammatory cyto-
kines IL-1?, IL-1?, and IL-12, were significantly higher in the
FIG 1 H5 HA MBS and virus replication in mice. Groups of female BALB/c
mice (n ? 4/group) were infected with 125 TCID50of either H5N1 (shaded
symbols) or ?H5N1 virus (open symbols) i.n., each symbol representing a
discrete data point in each group. Nasal turbinates, lungs, and the brain were
titer, expressed as log10TCID50/g of tissue. *, P ? 0.05 by Mann-Whitney U
Suguitan et al.
jvi.asm.orgJournal of Virology
lungs of mice infected with the H5N1 virus than in those infected
with the ?H5N1 virus, most notably on days 4 and 6 p.i. (Fig. 2).
IL-6, which were previously reported to be associated with HPAI
mice infected with either virus, perhaps reflecting the use of dif-
ferent assay systems with differing sensitivities of detection for
these cytokines. The concentrations of the chemokines monocyte
and KC (the murine equivalent of human IL-8) were also signifi-
cantly higher in the H5N1 virus-infected group (Fig. 2). These
chemokines are implicated in the recruitment of neutrophils and
monocytes in the pulmonary tissues of mice that result in acute
inflammation and severe lung pathology (31). The levels of the
anti-inflammatory cytokine IL-10 were also elevated, likely the
result of a negative feedback mechanism in response to the ele-
vated levels of inflammatory cytokines in the local milieu.
The H5 HA MBS is associated with lymphopenia in mice.
Tumpey et al. previously reported that an HPAI H5N1 infection
caused lymphopenia in mice (49). To assess the impact of the H5
HA MBS on the leukocyte population in the peripheral blood,
groups of mice were infected with either the H5N1 or ?H5N1
virus while another group was mock infected with medium to
serve as a control group. It is evident that mice infected with the
H5N1 virus had a higher proportion of neutrophils (Fig. 3A) and
monocytes (Fig. 3C) in the peripheral blood and a corresponding
animals, as early as day 1 p.i. Animals infected with the ?H5N1
virus exhibited a transient slight increase in the proportion of
tion of lymphocytes (Fig. 3B) early in infection but displayed a
WBC differential profile similar to that of the mock-infected
the H5N1 virus resulted in an alteration of the leukocyte popula-
tion in the peripheral blood of mice.
The H5 HA MBS and mouse lung pathology. Histopatholog-
ical analysis of lung tissue sections from ?H5N1-infected mice
showed minimal to mild focal inflammation of the large airways
by day 4 p.i. (Fig. 4A), progressing to moderate bronchial and
cleared by day 8 p.i. (data not shown). In contrast, there were
FIG 2 Cytokine and chemokine levels in the lungs of mice infected intranasally with the H5N1 or ?H5N1 virus. Groups of female BALB/c mice (n ? 4/group)
clarified lung homogenates from days 2, 4, 6, and 8 p.i. Cytokine concentrations are expressed as pg/ml. White bars represent mock-infected mice, gray bars
represent ?H5N1 virus-infected mice, and black bars represent H5N1 virus-infected mice. *, P ? 0.05 by Mann-Whitney U test.
H5 HA Cleavability and Pathogenicity in Mammals
March 2012 Volume 86 Number 5jvi.asm.org 2709
significant multifocal to locally extensive inflammation and ne-
crosis of the bronchial and bronchiolar epithelium, often accom-
panied by sloughing of the epithelium, in the lungs of H5N1-
macrophages and neutrophils admixed with cellular debris.
lar and peribronchiolar cuffing by lymphocytes, plasma cells, and
neutrophils were also noted. Thus, removal of the H5 HA MBS
large airways in the lungs, that was less severe than the lung pa-
thology caused by the H5N1 wt virus.
Virus replication in ferrets. We next examined the relevance
of the MBS to the virulence of H5N1 viruses in ferrets. The bio-
logical H5N1 (bH5N1) virus was included in this set of studies as
virus displayed pronounced morbidity, as evidenced by elevated
body temperatures, significant weight loss, and lethargy (data not
shown). Moreover, bH5N1- and rH5N1-infected ferrets showed
neurological symptoms such as hind limb paralysis, ataxia, and
convulsions requiring euthanasia on day 6 and day 8 p.i., respec-
tively. Ferrets infected with the ?H5N1 virus showed no clinical
signs of illness through 14 days p.i. The bH5N1, rH5N1, and
?H5N1 viruses all replicated to similar mean titers in the upper
respiratory tract of ferrets on days 3 and 6 p.i. (Fig. 5A). In the
lungs, the rH5N1 virus replicated to higher titers than did the
bH5N1 and ?H5N1 viruses on day 3 p.i. (Fig. 5B), but on day 6
p.i., most of the ferrets infected with the rH5N1 and bH5N1 vi-
ruses continued to display robust virus replication while virus
could no longer be detected in the lungs of ?H5N1-infected fer-
rets. The bH5N1 and rH5N1 viruses consistently disseminated to
and replicated in the brain while the replication of the ?H5N1
virus in the brain was severely restricted (Fig. 5C). Lung histology
findings within each animal and among each group ranged from
necrosis of alveolar walls with infiltrates of neutrophils and mac-
rophages in air spaces obliterating normal pulmonary architec-
of mice. Groups of female BALB/c mice (n ? 3/group) were infected i.n. with
125 TCID50of either H5N1 or ?H5N1 virus. WBC differential counts were
performed on peripheral blood smears that were prepared daily from day 1
through day 5 p.i. The percentage of each WBC type was based on counting at
least 100 WBCs under an oil-immersion objective.
FIG 4 Histopathological effects induced by the H5N1 virus in the lungs of
mice. Formalin-fixed, paraffin-embedded lung tissue sections from ?H5N1
virus (A and B)-, mock (C)-, or H5N1 virus (D, E, and F)-infected mice were
stained with hematoxylin and eosin. Representative sections from tissues har-
vested on days 4 (A, C, and D) and 6 (B, E, and F) p.i. are shown. Lungs from
bronchial and bronchiolar epithelium (D and E) with considerable sloughing
apparent (E), with multifocal thickening and/or obliteration of alveolar walls.
Lungs from ?H5N1-infected mice were minimally affected at day 4 (A) and
lium and variable peribronchiolar cuffing at day 6 (B).
Suguitan et al.
jvi.asm.org Journal of Virology
ture. There was no apparent correlation between the severity of
histological findings and virus titer (data not shown). These re-
sults indicate that the MBS is a virulence determinant in ferrets as
replication in the lower respiratory tract, restricted the spread of
the virus to extrapulmonary sites such as the brain, and was asso-
ciated with significant reductions in mortality and morbidity.
quality reagents specific to ferret proteins has hindered the devel-
opment of quantitative assays to determine the level of cytokines
ferrets. Instead, a quantitative real-time PCR (qPCR) was em-
and peripheral blood mononuclear cells (PBMCs) of ferrets in-
fected with the different H5N1 viruses, similar to the approach
most part, the bH5N1 and rH5N1 viruses induced similar cyto-
kine patterns, eliciting dramatically high mRNA levels for
CXCL10 and modest fold increases of the proinflammatory cyto-
kine IL-12 and several interferon-related genes such as MIG-1,
MX-1, OAS, and RIG-I (Fig. 6), while no significant fold differ-
ences in the mRNAs for IFN-?, IFN-?, or IFN-? were observed
(data not shown). There were also no significant differences ob-
for any of the H5N1 viruses evaluated (data not shown).
Virus replication and pathogenicity in AGMs. The replica-
green monkeys (AGMs; Chlorocebus aethiops), animals that are
mice and ferrets. After 2 ? 106TCID50of the H5N1 or ?H5N1
virus was administered i.n. and intratracheally to AGMs, they
were observed daily for evidence of clinical illness. Both viruses
geal swabs (Table 1). Virus replication was far more restricted in
the nasal turbinate tissue (Table 1); replication of the ?H5N1
virus was detected only on day 2 p.i. while the H5N1 virus was
detected at a low level until day 7 p.i. (Table 1). Neither virus was
viruses replicated to moderately high titers in the lungs (Table 1).
The H5N1 virus replicated to a slightly higher titer than did the
was undetectable. Interestingly, despite virus replication to about
104TCID50/g in the lungs, none of the AGMs infected with the
H5N1 virus experienced weight loss or displayed any clinical ill-
ness during the course of the study. There was also no apparent
difference in the histopathological analyses of lung tissue sections
of AGMs infected with the H5N1 or ?H5N1 virus or in the cyto-
kine profiles displayed by animals infected with either virus (data
H5 HA MBS in AGMs was earlier clearance of the virus from the
lungs. We were unable to assess the effect on extrapulmonary
spread because the H5N1 virus did not spread to the liver, spleen,
MBS motif at the HA cleavage site (52). Although the recon-
HA activation through a yet-undefined mechanism (48). The de-
letion of potential glycosylation sites and insertion of multiple
basic amino acid residues in the cleavage site of the HA of the
human influenza virus A/Aichi/2/1968 (H3N2) failed to confer
enhanced cleavability of the H3 HA to the extent seen in highly
pathogenic avian influenza viruses (22). Even when insertion of a
polybasic cleavage site in the HA of an H3N8 virus resulted in its
cleavability in the absence of exogenous trypsin and enhanced
replication in vivo, the modification was still not sufficient to ren-
FIG 5 The H5N1 virus replicated in the respiratory tract and brain of ferrets.
Groups of ferrets (n ? 4/group) were infected with 106TCID50of either
bH5N1 (gray squares), rH5N1 (black squares), or ?H5N1 virus (white
squares) i.n., each symbol representing a discrete data point in each group.
6 p.i. Horizontal bars represent the median virus titer, expressed as log10
TCID50/g of tissue.
H5 HA Cleavability and Pathogenicity in Mammals
March 2012 Volume 86 Number 5 jvi.asm.org 2711
der the virus highly pathogenic for poultry, indicating that a
highly cleavable HA is necessary but not sufficient to confer viru-
insertions in the connecting peptide of non-H5 or -H7 HAs likely
inhibit the generation of these types of viruses in nature (16).
an MBS in the HA protein is highly indicative of its cleavability
and is clearly associated with virulence in poultry, but the impact
of the H5 HA MBS on the virulence of HPAI H5N1 wt viruses in
mammals is unclear. While all H5N1 viruses that have infected
humans since 1997 possess the MBS in the H5 HA (1, 42), disease
associated with these infections ranges in severity from mild to
fatal, indicating possible contributions of other virus proteins to
virulence and/or the potential role of host factors in determining
FIG 6 Influence of H5 HA MBS on cytokine responses in ferrets. Groups of ferrets (n ? 3/group) were infected i.n. with 106TCID50of either bH5N1 (gray
squares), rH5N1 (black squares), or ?H5N1 virus (white squares). mRNA levels for various cytokines and immune-related genes from PBMCs isolated on day
calculated compared to preinfection levels of the same animals. Horizontal bars represent the median fold change difference for each group.
TABLE 1 The influence of the H5 HA MBS on the replication of the H5N1 virus in African green monkeysa
Mean virus titer ? SE (log10TCID50/ml org) in tissue samples collected on indicated day p.i.:
Day 1 Day 2Day 3Day 4Day 7
Nasopharyngeal swab H5N1
3.6 ? 0.5
2.5 ? 0.8
2.6 ? 0.5
1.8 ? 0.6
2.3 ? 0.3
2.0 ? 0.5
4.0 ? 0.6
3.6 ? 0.5
1.7 ? 0.7
0.8 ? 0.1
1.6 ? 0.5
1.4 ? 0.8
2.0 ? 0.5
4.1 ? 0.3
3.0 ? 0.4
2.4 ? 0.9
0.8 ? 0.3
2.3 ? 0.8
2.4 ? 0.2
aAGMs were infected with 2 ? 106TCID50of either the H5N1 or ?H5N1 virus i.n. and intratracheally. Nasal swabs were collected on the indicated days while nasal turbinates and
lungs were harvested on days 2, 4, and 7 p.i. Virus titers are expressed as mean ? standard error log10TCID50/g of tissue or per ml of swabs. ND, not determined.
bLower limit of detection.
Suguitan et al.
jvi.asm.org Journal of Virology
disease susceptibility. Chen et al. (2007) investigated virulence
determinants of the 1997 Hong Kong H5N1 wt viruses and con-
the polymerase basic protein 2 (PB2), of these viruses make inde-
pendent contributions to virulence (8). The E627K mutation in
PB2 has been implicated in the systemic dissemination and viru-
host immune response in mice (12), but this mutation is not es-
sential for virulence in ferrets (14, 28) or in humans (11). The
contribution of the other polymerase proteins to the virulence of
H5N1 viruses in ferrets and mice has also been reported (10, 13,
33, 35, 38), demonstrating the complex interplay among various
viral gene products that determine the virulence of H5N1 viruses.
In order to control for virulence determinants in other gene
segments, we used two isogenic recombinant H5N1 wt viruses
HA protein. When all other genetic characteristics of the HPAI
A/Vietnam/1203/2004 (H5N1) wt virus were kept constant, the
effect of the H5 HA MBS on virulence depended on the host.
nonstructural proteins, the H5N1 and ?H5N1 viruses differed in
their replication efficiency in vivo, and this could have affected
their ability to induce proinflammatory cytokines and modulate
the immune response in specific mammalian hosts. In mice, the
ity of the H5N1 wt virus (15, 43). This is not too surprising given
that the H5 HA MBS was associated with enhanced virus replica-
tion, systemic virus dissemination, and lymphopenia. Mice in-
fected with the H5N1 virus had significantly elevated levels of
inflammatory cytokines and chemokines in the lungs, associated
with infiltration of macrophages and neutrophils. The recruit-
ment of these cells to the lungs likely contributes to acute lung
inflammation and severe lung pathology (31).
inducing fever, significant weight loss, lethargy, and neurological
symptoms. The H5N1 virus displayed enhanced magnitude and
analysis, Cameron et al. (2008) showed that ferrets experience a
by upregulation of interferon response genes and robust expres-
sion of the inflammatory chemokine CXCL10 in the lungs upon
H5N1 virus infection (7). Our data lend support to this report:
elevated mRNA levels of CXCL10, as well as several interferon
response genes, were detected in the peripheral blood of H5N1-
attachment of avian influenza viruses as do humans (51), and the
clinical illness associated with H5N1 influenza virus infection in
55), although diarrhea, neurologic symptoms, and systemic dis-
semination are not commonly seen in human H5N1 cases.
In nonhuman primates, the replication of both the H5N1 and
?H5N1 viruses in the respiratory tract of AGMs was modest and
neither virus caused clinical symptoms. The H5N1 virus repli-
cated to a slightly higher titer than did the ?H5N1 virus in the
lungs. The low virulence of H5N1 virus in AGMs contrasts with
reports of severe disease in cynomolgus macaques (Macaca fas-
cicularis), where alveolar and bronchiolar lesions were apparent
innate immune gene expression was observed (2). Host genetic
differences between these two species of nonhuman primates
might explain the difference in their susceptibilities to HPAI
in AGMs and the absence of clinical disease suggest that AGMs
may reflect milder forms of human disease caused by avian influ-
respiratory distress syndrome and multiorgan failure. AGMs are
The recent identification and elucidation of human cellular
factors and the signaling pathways that are crucial for influenza
virus replication (21, 26, 34) highlight the role that host genetics
Different inbred laboratory strains of mice differ in their kinetics
H1N1 virus, suggesting that the host genetic background has a
great influence on the immune response to influenza virus infec-
tion (36), and genetic elements that map to different loci of the
mouse genome have been associated with resistance to H5N1 vi-
rus infection (4). In contrast to mice, ferrets and AGMs are out-
bred animals and this is reflected in the variability in the data.
In summary, when all the genetic components of an HPAI
H5N1 wt virus are kept equal, the MBS of the H5 HA protein acts
as a virulence marker in a host-specific manner in mammals. The
H5 HA MBS was associated with lethality, enhanced virus repli-
cation, and systemic dissemination in mice and ferrets. In AGMs,
it was associated with mild enhancement of replication and de-
layed clearance from the respiratory tract. Thus, the contribution
of H5 HA MBS to the virulence of the H5N1 HPAI virus varies
less remarkable in nonhuman primates, underscoring the role of
host genetic differences in the susceptibility of mammals to HPAI
for superior technical support for the animal studies conducted at the
ies in ferrets and AGMs at Bioqual. We are grateful to Hong Jin and
Institute of Hygiene and Epidemiology (NIHE), Vietnam, for providing
the A/Vietnam/1203/2004 (H5N1) virus used in this study, which was
made available to us by Nancy Cox and Alexander Klimov, Influenza
Division, Centers for Disease Control and Prevention (CDC), Atlanta,
GA. We also thank David Tabor, Kathy Wang, and Xiamou Chen of
MedImmune for their assistance and support in conducting the qPCR
assays for ferret cytokine and chemokine genes.
1. Abdel-Ghafar AN, et al. 2008. Update on avian influenza A (H5N1) virus
infection in humans. N. Engl. J. Med. 358:261–273.
2. Baskin CR, et al. 2009. Early and sustained innate immune response
defines pathology and death in nonhuman primates infected by highly
pathogenic influenza virus. Proc. Natl. Acad. Sci. U. S. A. 106:3455–3460.
3. Bhatia M, Moochhala S. 2004. Role of inflammatory mediators in the
pathophysiology of acute respiratory distress syndrome. J. Pathol. 202:
4. Boon AC, et al. 2009. Host genetic variation affects resistance to infection
with a highly pathogenic H5N1 influenza A virus in mice. J. Virol. 83:
H5 HA Cleavability and Pathogenicity in Mammals
March 2012 Volume 86 Number 5jvi.asm.org 2713
5. Bosch FX, Garten W, Klenk HD, Rott R. 1981. Proteolytic cleavage of Download full-text
influenza virus hemagglutinins: primary structure of the connecting pep-
genicity of avian influenza viruses. Virology 113:725–735.
6. Bosch FX, Orlich M, Klenk HD, Rott R. 1979. The structure of the
hemagglutinin, a determinant for the pathogenicity of influenza viruses.
7. Cameron CM, et al. 2008. Gene expression analysis of host innate
immune responses during lethal H5N1 infection in ferrets. J. Virol. 82:
8. Chen H, et al. 2007. Polygenic virulence factors involved in pathogenesis
of 1997 Hong Kong H5N1 influenza viruses in mice. Virus Res. 128:159–
9. Chen H, et al. 2006. Establishment of multiple sublineages of H5N1
influenza virus in Asia: implications for pandemic control. Proc. Natl.
Acad. Sci. U. S. A. 103:2845–2850.
10. Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P. 2007. A
single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A
viruses contributes to increased virulence. PLoS Pathog. 3:1414–1421.
11. de Jong MD, et al. 2006. Fatal outcome of human influenza A (H5N1) is
12. Fornek JL, et al. 2009. A single-amino-acid substitution in a polymerase
protein of an H5N1 influenza virus is associated with systemic infection
and impaired T-cell activation in mice. J. Virol. 83:11102–11115.
the transmission of H5N1 avian influenza viruses in a mammalian host.
PLoS Pathog. 5:e1000709.
14. Govorkova EA, et al. 2005. Lethality to ferrets of H5N1 influenza viruses
isolated from humans and poultry in 2004. J. Virol. 79:2191–2198.
15. Hatta M, Gao P, Halfmann P, Kawaoka Y. 2001. Molecular basis for high
virulence of Hong Kong H5N1 influenza A viruses. Science 293:1840–
16. Horimoto T, Kawaoka Y. 1997. Biologic effects of introducing additional
avian influenza virus. Virus Res. 50:35–40.
17. Horimoto T, Kawaoka Y. 1994. Reverse genetics provides direct evidence
for a correlation of hemagglutinin cleavability and virulence of an avian
influenza A virus. J. Virol. 68:3120–3128.
18. Horimoto T, Nakayama K, Smeekens SP, Kawaoka Y. 1994. Proprotein-
processing endoproteases PC6 and furin both activate hemagglutinin of
virulent avian influenza viruses. J. Virol. 68:6074–6078.
19. Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA. 2008. A new
influenza virus virulence determinant: the NS1 protein four C-terminal
20. Kandun IN, et al. 2006. Three Indonesian clusters of H5N1 virus infec-
tion in 2005. N. Engl. J. Med. 355:2186–2194.
21. Karlas A, et al. 2010. Genome-wide RNAi screen identifies human host
factors crucial for influenza virus replication. Nature 463:818–822.
22. Kawaoka Y. 1991. Structural features influencing hemagglutinin cleav-
ability in a human influenza A virus. J. Virol. 65:1195–1201.
23. Kawaoka Y, Webster RG. 1989. Interplay between carbohydrate in the
stalk and the length of the connecting peptide determines the cleavability
of influenza virus hemagglutinin. J. Virol. 63:3296–3300.
24. Kawaoka Y, Webster RG. 1988. Sequence requirements for cleavage
Proc. Natl. Acad. Sci. U. S. A. 85:324–328.
25. Klenk HD, Rott R, Orlich M, Blodorn J. 1975. Activation of influenza A
viruses by trypsin treatment. Virology 68:426–439.
26. Konig R, et al. 2010. Human host factors required for influenza virus
replication. Nature 463:813–817.
27. Maeda T, Ohnishi S. 1980. Activation of influenza virus by acidic media
causes hemolysis and fusion of erythrocytes. FEBS Lett. 122:283–287.
28. Maines TR, et al. 2005. Avian influenza (H5N1) viruses isolated from
humans in Asia in 2004 exhibit increased virulence in mammals. J. Virol.
29. Maines TR, et al. 2008. Pathogenesis of emerging avian influenza viruses
in mammals and the host innate immune response. Immunol. Rev. 225:
30. Nicholls JM, et al. 2007. Tropism of avian influenza A (H5N1) in the
upper and lower respiratory tract. Nat. Med. 13:147–149.
31. Perrone LA, Plowden JK, Garcia-Sastre A, Katz JM, Tumpey TM. 2008.
H5N1 and 1918 pandemic influenza virus infection results in early and
PLoS Pathog. 4:e1000115.
32. Rott R, Klenk HD, Nagai Y, Tashiro M. 1995. Influenza viruses, cell
enzymes, and pathogenicity. Am. J. Respir. Crit. Care Med. 152:S16–S19.
33. Salomon R, et al. 2006. The polymerase complex genes contribute to the
high virulence of the human H5N1 influenza virus isolate A/Vietnam/
1203/04. J. Exp. Med. 203:689–697.
34. Shapira SD, et al. 2009. A physical and regulatory map of host-influenza
interactions reveals pathways in H1N1 infection. Cell 139:1255–1267.
35. Song MS, et al. 2009. The polymerase acidic protein gene of influenza a
virus contributes to pathogenicity in a mouse model. J. Virol. 83:12325–
36. Srivastava B, et al. 2009. Host genetic background strongly influences the
response to influenza A virus infections. PLoS One 4:e4857.
37. Stech O, et al. 2009. Acquisition of a polybasic hemagglutinin cleavage
site by a low-pathogenic avian influenza virus is not sufficient for imme-
diate transformation into a highly pathogenic strain. J. Virol. 83:5864–
38. Steel J, Lowen AC, Mubareka S, Palese P. 2009. Transmission of influ-
enza virus in a mammalian host is increased by PB2 amino acids 627K or
627E/701N. PLoS Pathog. 5:e1000252.
39. Steiner DF, Smeekens SP, Ohagi S, Chan SJ. 1992. The new enzymology
of precursor processing endoproteases. J. Biol. Chem. 267:23435–23438.
40. Steinhauer DA. 1999. Role of hemagglutinin cleavage for the pathogenic-
ity of influenza virus. Virology 258:1–20.
41. Stieneke-Grober A, et al. 1992. Influenza virus hemagglutinin with mul-
tibasic cleavage site is activated by furin, a subtilisin-like endoprotease.
EMBO J. 11:2407–2414.
42. Subbarao K, et al. 1998. Characterization of an avian influenza A (H5N1)
43. Suguitan AL, Jr, et al. 2009. The influence of the multi-basic cleavage site
of a live attenuated influenza A H5N1 cold-adapted vaccine virus. Virol-
44. Suguitan AL, Jr, et al. 2006. Live, attenuated influenza A H5N1 candidate
vaccines provide broad cross-protection in mice and ferrets. PLoS Med.
45. Suzuki Y, et al. 2000. Sialic acid species as a determinant of the host range
of influenza A viruses. J. Virol. 74:11825–11831.
predict morbillivirus disease outcome in ferrets. Virology 362:404–410.
47. Szretter KJ, et al. 2007. Role of host cytokine responses in the pathogen-
esis of avian H5N1 influenza viruses in mice. J. Virol. 81:2736–2744.
48. Tumpey TM, et al. 2005. Characterization of the reconstructed 1918
Spanish influenza pandemic virus. Science 310:77–80.
49. Tumpey TM, Lu X, Morken T, Zaki SR, Katz JM. 2000. Depletion of
lymphocytes and diminished cytokine production in mice infected with a
highly virulent influenza A (H5N1) virus isolated from humans. J. Virol.
50. Ungchusak K, et al. 2005. Probable person-to-person transmission of
avian influenza A (H5N1). N. Engl. J. Med. 352:333–340.
cells in the lower respiratory tract of humans and other mammals. Am. J.
52. Webster RG, Rott R. 1987. Influenza virus A pathogenicity: the pivotal
role of hemagglutinin. Cell 50:665–666.
53. White J, Matlin K, Helenius A. 1981. Cell fusion by Semliki Forest,
influenza, and vesicular stomatitis viruses. J. Cell Biol. 89:674–679.
to WHO. World Health Organization, Geneva, Switzerland. http://www
55. Zitzow LA, et al. 2002. Pathogenesis of avian influenza A (H5N1) viruses
in ferrets. J. Virol. 76:4420–4429.
Suguitan et al.
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