Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase.
ABSTRACT Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.
We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity.
IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM.
IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.
- SourceAvailable from: Kridsada Chaichoun[Show abstract] [Hide abstract]
ABSTRACT: To characterise neuraminidase (NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus.World journal of virology. 11/2014; 3(4):30-6.
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ABSTRACT: Despite the availability of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to have a significant, annual impact on the morbidity and mortality of human beings, highlighting the continued need for research in the field. Current vaccine strategies predominantly focus on raising a humoral response against hemagglutinin (HA)-the more abundant, immunodominant glycoprotein on the surface of the influenza virus. In fact, anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. Yet, the quest to discern the exact importance of NA-based protection is decades old. Also, while antibodies against the NA glycoprotein fail to prevent infection of the influenza virus, anti-NA immunity has been shown to lessen the severity of disease, decrease viral lung titers in animal models, and reduce viral shedding. Growing evidence is intimating the possible gains of including the NA antigen in vaccine design, such as expanded strain coverage and increased overall immunogenicity of the vaccine. After giving a tour of general influenza virology, this review aims to discuss the influenza A virus neuraminidase while focusing on both the historical and present literature on the use of NA as a possible vaccine antigen.Viruses 06/2014; 6(6):2465-2494. · 3.28 Impact Factor
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RESEARCH Open Access
Influenza A penetrates host mucus by cleaving
sialic acids with neuraminidase
Miriam Cohen1, Xing-Quan Zhang2, Hooman P Senaati1, Hui-Wen Chen2,3, Nissi M Varki1, Robert T Schooley2
and Pascal Gagneux1*
Background: Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with
oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of
NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected
cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus
layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the
cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated
decoys, but the exact mechanism is not yet established.
Methods: We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections,
and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The
protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia
content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used
to quantify NA activity.
Results: IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent
manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells.
HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from
HSM but not from PSM, and binds to HSM but not to PSM.
Conclusion: IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV
infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus
NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a
promising new avenue for drug development.
Keywords: Influenza A, Sialic acids, Mucus, Neuraminidase, Infection, Saliva
Interactions of Influenza A viruses (IAVs) with mucus
were first described in the mid 20thcentury, and led early
researchers to classify influenza as a (ortho)myxovirus – a
virus with affinity for mucus [1,2]. IAVs must penetrate a
secreted mucus layer (up to 50 μm thick) to reach target
tissues in mammalian airways . Mucus is a defensive
layer containing highly glycosylated mucins rich in ter-
minal sialic acids (Sias) . It has been suggested that
mucus may protect against IAV infection by presenting
sialylated “decoys” that mimic receptors on the cell surface
[5,6]. Influenza viruses bind these unproductive receptors,
become trapped in the mucus layer, and can then be
removed by the normal process of mucus clearance as part
of the innate defense system [7,8].
Neuraminidase (NA) enzymatic activity cleaves Sias
from glycoconjugates on infected cells and progeny vi-
rions allowing budding virions to escape from infected
cells. NA inhibitors such as oseltamivir inhibit viral re-
lease by preventing the cleavage of Sias specifically .
It has also been shown that NA inhibition with oselta-
mivir carboxylate suppresses IAV infection of cultured
* Correspondence: email@example.com
1Department of Cellular and Molecular Medicine, University of California San
Diego, 9500 Gilman Dr, La Jolla 92093, California, USA
Full list of author information is available at the end of the article
© 2013 Cohen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication
waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise
Cohen et al. Virology Journal 2013, 10:321
human airway epithelium (HTBE) . Thus NA activity
must have a role during initial infection, but the exact
mechanism is not yet known. Since HTBE cells are coated
with a secreted mucus layer, it was speculated that NA
activity promotes infection by cleaving Sias from mucus
. However, other interpretations such as promotion
of hemagglutinin-mediated fusion [11,12] cannot yet be
ruled out, and IAV interactions with mucins during initial
infection remain poorly characterized.
Mucin glycosylation and sialylation vary significantly
between species, and thus could influence influenza host
species specificity. Human airway and salivary mucins
have been well characterized [13-15] and their constitu-
ents and glycosidic linkages differ dramatically from those
of other species such as chimpanzees  and pigs .
Humans express predominantly N-acetylneuraminic acid
(Neu5Ac) . Human airway sialoglycans lack the sialic
acid (Sia) N-glycolylneuraminic acid (Neu5Gc), which is
the predominant Sia in porcine mucus . In the human
upper respiratory tract Sias are predominantly found in
α2-6 glycosidic linkage . In contrast, these Sias are
mostly α2-3-linked in chimpanzees  and pigs .
Mucus carries a vast array of variable receptors, and
mucin sialylation can create sites in the mucus that resem-
ble the target receptors on cells [16,21]. IAV binding prop-
erties are important determinants of host susceptibility
and host range, and the type and distribution patterns of
sialylated glycans on target tissues seem to be crucial .
Notably, binding properties of IAV are traditionally stud-
ied in absence of secreted mucins despite the fact that all
natural infection sites are abundantly covered with these
In this study we investigate the interaction of IAV with
mucins, and provide experimental evidence for the role
of NA during initial infection of mucus-coated cells and
tissues. We demonstrate Sia-dependent binding of IAV
to secreted mucus on frozen human trachea/bronchus
tissues. We present direct in vitro evidence that secreted
mucus protects underlying cells from infection by present-
ing sialylated decoys for hemagglutinin (HA) and compet-
ing for NA cleavage activity. We used purified mucus
from two different hosts: human and pig, to show direct
cleavage of- and direct binding to- sialylated human
mucus by human IAV NA and HA, respectively.
Influenza A virus interacts with mucin on human airway
IAV tropism depends on HA binding specificity and the
host sialylation pattern. The distribution of terminal Sias
in α2-6 and in α2-3 linkages varies along the respiratory
tract, and changes with age and developmental stage
[19,23]. Human respiratory tract sialylation patterns have
been extensively studied on paraffin embedded tissues,
which are lacking much of the secreted mucus layer
[23,24]. Here we examine glycosylation and IAV binding
to frozen human trachea/bronchus tissues that were fro-
zen and embedded in optimal cutting temperature (OCT)
compound. This treatment preserves the secreted mucus
layer in a natural state, enabling both immunohistochem-
istry and virus binding studies . Secreted mucus forms
a visible lining on the epithelium of human bronchial tis-
sues, detected by Periodate Acid Schiff staining (Figure 1,
PAS, dashed line indicates secreted mucus). Potential
receptors for human IAV on secreted airway mucus were
detected with Sambucus nigra lectin (SNA), which binds
to Siaα2-6Gal/GalNAc, or with TKH2 antibody, which
bind to Siaα2-6GalNAc on O-linked glycans (Sialyl Tn)
(Figure 1, SNA & TKH2, outlined dark brown staining).
Sialyl Tn is a glycan epitope that is abundant on mucins
but infrequent in other tissues . TKH2 staining is con-
fined to the secreted material lining the epithelium and
the glands (Figure 1, TKH2), further confirming that this
material represents the secreted mucus layer. In order to
test the ability of IAV to bind secreted mucus, these tis-
sues were incubated with 600 HAU of two seasonal virus
strains, A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2), and
a clinical isolate of the pandemic A/SD/1/2009(SOIV). All
three virus strains bound to secreted mucus as well as to
the underlying ciliated cells (Figure 1, lower panels, dashed
lines). Removal of Sias from the tissues by enzymatic
cleavage with Arthrobacter ureafaciens sialidase (Figure 2,
AUS) significantly reduces virus binding to the mucus,
confirming specific binding to sialylated receptors.
Similarly, truncation of the Sia side chain by mild so-
dium periodate treatment  reduces virus binding to
the mucus (Figure 2, NaIO4). These findings confirm
that the secreted mucus layer presents sialylated decoy
receptors for binding by IAV and other pathogens.
Human mucin protects cells from infection in vitro
Since IAV can both bind and cleave sialylated epitopes, we
tested whether sialylated mucins can effectively protect
underlying cells from IAV infection in vitro. Confluent
monolayers of MDCK cells in 16-well Lab-Tek chamber
slides were overlaid with human salivary mucins (HSM),
porcine submaxillary mucins (PSM) or buffer. The mucin
content of HSM is similar to that of human airway epithe-
lium submucosal glands (Additional file 1A) . HSM
preparation is enriched by acid precipitation of mucins
and filtration of saliva samples  (Additional file 1B-C).
Thus the HSM preparation is a good representation of the
mucus of human upper respiratory system. The Sia con-
tents of HSM and PSM samples were determined by
DMB-HPLC (Additional file 1D), and MDCK cells were
overlaid with mucus containing a known amount of Sias.
Total Sia content in the wells was 12,000 pmol/well (high),
3,200 pmol/well (medium) or 1,500 pmol/well (low). The
Cohen et al. Virology Journal 2013, 10:321
Page 2 of 13
protection efficacy of HSM and PSM against IAV infection
of the underlying cells was determined by challenging the
cells with 109TCID50 of four IAV strains: A/PR/8/34
(H1N1), A/SD/1/2009(SOIV), A/SD/17/2008(H1N1), and
A/Aichi/2/68(H3N2) for 1 h at 37°C. Cells were then
washed to remove both virus and mucus, and fresh
DMEM-TPCK media was added. The cells were incubated
for additional 5.5 h at 37°C, fixed and stained for viral nu-
clear proteins. The number of infected cells was quantified
in twenty randomly selected images from each sample,
and the infection rate relative to buffer coated cells was
determined (Figure 3). For all four IAV strains, coating of
cells with HSM at medium or high Sia content signifi-
cantly reduces the infection of underlying cells compared
to buffer coated cells (Figure 3, compare PBS (0) to HSM
(3200) and HSM (12000 pmol), P<0.05). Only three of the
tested IAV strains were significantly inhibited by HSM at
low Sia content: A/PR/8/34(H1N1), A/SD/17/2008(H1N1)
and A/Aichi/2/68(H3N2) (Figure 3A and C-D, compare
PBS (0) to HSM (1500 pmol), P<0.05). Notably a dose
effect of Sia-content in the HSM layer was observed for
three of the IAV strains (A/PR/8/34(H1N1), A/SD/2009
(SOIV) and A/Aichi/2/68(H3N2)) where higher Sia-
content resulted in fewer infected cells. The numbers
of infected cells were 60-95% lower in monolayers
coated with HSM (high), 40-65% lower in monolayers
coated with HSM (medium), and 40-50% lower in
monolayers coated with HSM (low), depending on the
strain (Figure 3A-B and D, see Additional file 2 for
complete statistical analysis). In contrast to HSM,
Figure 1 IAV binds to secreted mucus in human trachea tissues. Frozen human trachea tissue sections were stained with Hematoxylin and
Eosin (H&E), periodic acid Schiff (PAS, mucin staining in pink), Sambucus nigra agglutinin (SNA, binds to Siaα2-6Gal/GalNAc) or TKH2 antibody
(binds to Sialyl Tn: Siaα2-6GalNAc on O-linked glycans). Dashed lines specify the location of secreted mucus, which is preserved in the frozen
tissues, as seen in H&E and PAS staining. Both SNA and TKH2 bound to the secreted mucus (dark brown staining), indicating abundant potential
ligands for IAV binding. TKH2 binding was confined to the lining of the epithelium and the glands, further confirming that the secreted mucus
layer is adequately preserved in these tissues. Binding of IAV to the tissues was tested by incubating 600 HAU of virus 1.5 h at room temperature.
All three strains bound to the secreted mucus layer (dashed line), and to ciliated cells (cells stained in dark brown). Virus was detected by anti-NP
antibodies. Boxed area is enlarged below each image. Scale bar indicates 500 μm, scale bar of enlarged area indicates 50 μm.
Cohen et al. Virology Journal 2013, 10:321
Page 3 of 13
coating cells with PSM typically did not result a significant
reduction in the number of infected cells (Figure 3A-B
and D). However, a mild reduction (15-25%) in number of
cells infected by A/SD/17/2008(H1N1) was observed in
PSM coated monolayers (Figure 3C, P<0.05).
High Sia-content PSM (high) is comprised of 10,200
pmol Neu5Gc and 1,800 pmol Neu5Ac (Additional
file 1D). However, the presentation of Sia differs between
PSM  and HSM [13-15]. Despite having a similar
Neu5Ac content, PSM (high) and HSM (low) did not have
the same inhibitory effect on IAV infection. Two strains,
A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) were not sig-
nificantly inhibited by coating monolayers with PSM
(high), however coating monolayers with HSM (low) sig-
nificantly reduced the number of infected cells (Figure 3A
and D, P<0.0005, and P<0.0462, respectively). Numbers of
cells infected by A/SD/17/2008(H1N1) were reduced in
monolayers coated with both PSM (high) and HSM
(low), however, fewer infected cells were observed in
HSM-coated monolayers compared with PSM-coated
monolayers (Figure 3C, P<0.0504). In contrast coating
monolayers with either PSM (high) or HSM (low) did
not significantly reduce the number of cells infected
with A/SD/1/2009(SOIV) (Figure 3B). Thus Sia content,
type, and presentation are all important factors for
inhibition of IAV infection. Furthermore, mucus inhib-
ition of IAV infection is strain-dependent.
Inhibition of IAV neuraminidase by oseltamivir increases
the protective effect of HSM but not PSM
Since the virus NA can potentially cleave sialylated recep-
tors presented on secreted human mucus, inhibition of
NA activity may enhance the protective effect of mucus.
In order to test this hypothesis, 1 μM oseltamivir was
Figure 2 IAV binding to secreted mucus is Sia-dependent. Human trachea tissue sections were treated with Arthrobacter ureafaciens sialidase
(AUS), which cleaves Sias, or with mild sodium periodate (NaIO4), which truncates the Sia side chain. Both treatments reduce IAV binding to the
secreted mucus on human trachea tissues compared to untreated control tissues, confirming that IAV binding to the secreted mucus is Sia-dependent.
Dashed lines specify location of virus binding to secreted mucus. Scale bar indicates 50 μm.
Cohen et al. Virology Journal 2013, 10:321
Page 4 of 13
added to IAV prior to challenging the mucus-coated cells.
The most prominent reduction of infection rate by oselta-
mivir was observed for the pandemic A/SD/2009(SOIV)
strain. A reduction of ~60% in the number of infected
cells was observed in monolayers coated with either buffer
or PSM (Figure 3B, PBS and PSM P<0.05). This could
be attributed to oseltamivir inhibition of the secondary
Sia-binding site found on the virus N1 neuraminidase
Figure 3 HSM and oseltamivir have additive inhibitory effects. MDCK cells were layered with PSM or HSM at 1,500, 3,200, 12,000 pmol Sia/
well or with PBS buffer as control. The cells were challenged for 1h at 37°C with 109TCID50of (A) A/PR/8/34(H1N1), (B) A/SD/1/2009(H1N1),
(C) A/SD/17/2008(H1N1), or (D) A/Aichi/2/68(H3N2) in the presence (gray bars) or absence (black bars) of 1 μM oseltamivir. Infected cells were
identified by staining with anti-NP antibodies, and quantified in twenty randomly selected images from each sample. Experiments were repeated
three times, for each experiment the number of infected cells in the PBS-coated sample was set to 1, and the relative number of infected cells
for each treatment was calculated. Lower number of infected cells was observed in HSM coated monolayers compared to PBS-coated monolayers
for all tested virus strains (A-D, P<0.05). Dose effects of Sia content in HSM-coated samples were observed for three IAV strains (A-B, D). Significant
reduction in the number of infected cells in PSM-coated monolayers was observed only for one strain (C, P<0.05). With exception of the A/SD/1/2009
(H1N1) strain, oseltamivir did not inhibit infection of cell coated with either PBS or PSM (A, C-D, gray bars). In contrast, addition of oseltamivir
to HSM-coated samples further reduced infection of A/PR/8/34(H1N1), A/SD/1/2009(H1N1) strains (A-B). Both of the oseltamivir-insensitive
strains, A/SD/17/2008(H1N1) and A/Aichi/2/68(H3N2), were not affected by addition of the drug (C-D). Data was analyzed by 3-way ANOVA,
corrected for multiple comparisons using Tukey’s HSD (see Additional file 2 for complete statistical analysis data). Error bars represent standard
deviation. *P<0.05, **P<0.005.
Cohen et al. Virology Journal 2013, 10:321
Page 5 of 13
, rather than to the neuraminidase enzymatic activity.
However, the number of infected cells in HSM-coated
monolayers was further reduced to <10% upon addition of
oseltamivir, even with monolayers that were coated with
HSM (low). Thus oseltamivir and HSM have an additive
inhibitory effect (HSM + oseltamivir compared with HSM,
or with PBS + oseltamivir, P<0.05, see Additional file 2 for
complete statistical analysis). The number of infected
cells in PSM (high)-coated monolayers and HSM (low)-
coated monolayers were reduced to a similar extent by
the addition of 1 μM oseltamivir (Figure 3B, 24±8% and
11±7%, respectively, P<0.0853).
Similar results were obtained for the seasonal H1N1
strain (A/PR/8/34(H1N1)). Although the number of in-
fected cells in PSM- or PBS-coated monolayers was
not significantly reduced by oseltamivir, 25-30% reduction
in the number of infected cells was observed (Figure 3A,
gray bars). However, the number of infected cells in HSM-
coated monolayers was further reduced to <5% even in
monolayers coated with only a low Sia-content HSM
(Figure 3A, gray bars, P<0.05).
The clinical isolate A/SD/17/2008(H1N1) contains the
oseltamivir-resistant mutation H275Y in the NA gene, as
determined by cDNA sequencing (data not shown). An
overall reduction of 12-44% in the number of cells infected
by this strain was observed in oseltamivir-supplemented
samples (Figure 3C, compare gray and black bars for each
treatment). However, this effect was mostly not statistically
significant, and all of the oseltamivir-supplemented sam-
ples had similar number of infected cells regardless of the
mucus content (Figure 3C, compare all gray bars). Thus,
the oseltamivir effect seen with this strain is likely attrib-
uted to inhibition of a secondary Sia binding site of N1
neuraminidase. In contrast, infection with A/Aichi/2/68
(H3N2) strain was not affected by addition of 1 μM oselta-
mivir (Figure 3D, gray bars).
Taken together this suggests that NA sialidase activity
is important to release the virus from the HSM layer.
Direct evidence for cleaving of Sias by viral Neuraminidase
In order to demonstrate that our viruses can cleave Sias
from HSM, IAV was incubated with HSM and PSM
conjugated magnetic beads. HSM and PSM were
biotinylated and captured in streptavidin magnetic
beads. As a control, biotinylated polyacrylamide-Galβ1-
3GalNAc (T antigen) conjugated beads were also pre-
pared. Beads were incubated with 50 μl A/PR/8/34
(H1N1) (2048 HAU), A/Aichi/2/68(H3N2) (600 HAU),
or DMEM-TPCK buffer for 1.5 h at room temperature
to allow cleavage of sialylated beads. The beads were
then extensively washed to remove both virus and
cleaved (released) Sias molecules, and were then fixed
with formalin. Sia content of the beads was analyzed by
DMB-HPLC (Figure 4). Neu5Ac comprises 100% of the
Sias in HSM samples, in contrast, Sias from PSM
samples consist of ~30% Neu5Ac and 70% Neu5Gc
(Additional file 1D). Both virus strains cleaved Neu5Ac
from HSM, reducing the total Sia content by 40-60%
compared to beads incubated with buffer alone (Figure 4,
P<0.001). In contrast, cleavage of Sias from PSM coated
beads was less effective. Both virus strains reduce Neu5Ac
content by 15-23% (Figure 4, P<0.012), and only A/Aichi/
2/68(H3N2) cleaved Neu5Gc (Figure 4, hatched bars,
P=0.03). To our knowledge, this is the first direct dem-
onstration that NA can cleave Sias from mucus, and we
show that IAV effectively cleaves Neu5Ac from HSM
but is ineffective at cleaving Sias from PSM.
Cleaving specificity of viral neuraminidase
The cleaving preference for Sia type was tested for six
IAV strains using different substrates: Neu5Ac, Neu5Gc,
and 2-keto-3-deoxynononic acid (Kdn), each linked to
the fluorescent reporter 4-methyl-umbelliferyl (4MU,
Figure 5) . Virus (32-64 HAU) was diluted 10-fold
in MES buffer and incubated with 4MU-Sia substrates
(0-5,000 pmol) for 1 h at 37°C. The enzymatic activity
of each virus NA was determined by quantifying the
release of fluorescent 4MU compound. To account for
spontaneous release of 4MU due to instability of the
4MU-Sia compounds (see Additional file 3), MES buffer
was added instead of virus. Fluorescence in these sam-
ples was deemed background. As expected, all of the
tested virus strains cleaved Neu5Ac (Figure 5A, black
Figure 4 IAV effectively cleaves sialylated HSM. Magnetic
beads-conjugated to HSM or PSM were incubated with A/PR/8/34
(H1N1), A/Aichi/2/68(H3N2), or buffer at room temperature to allow
cleavage of sialylated beads. After 1.5 h incubation the Sia content
of the beads was analyzed by DMB-HPLC, and is expressed as
percent of Sia content in buffer-incubated beads. Solid bars represent
Neu5Ac content, and hatched bars represent Neu5Gc content. Both
viruses reduce Neu5Ac content of HSM by 40%-60%, in contrast, only
mild cleavage of PSM Sias was observed. ***P<0.001, **P<0.012
*P=0.03 values indicate the significance in difference between Sia
content in the virus-treated samples and the corresponding buffer
control (two-tailed T-Test, n=3).
Cohen et al. Virology Journal 2013, 10:321
Page 6 of 13
diamonds), and did not cleave Kdn (Figure 5A, black
circles), which typically is not found in this unmodified
form on mammalian tissues [31,32]. Interestingly, all six
viral strains cleaved Neu5Gc as well, although to lesser
extent than Neu5Ac (Figure 5A, black squares). This is
surprising since the same IAV strains were ineffective at
cleaving Neu5Gc from PSM (Figure 4). The viruses
showed different susceptibility to inhibition by oseltamivir.
Enzymatic activity of A/PR/8/34(H1N1), A/Denver/1/
57(H1N1), and A/SD/1/2009(SOIV) was abolished by
addition of 1 μM oseltamivir (Figure 5A and B, top
panels). As expected, A/SD/17/2008(H1N1) and A/SD/
21/2008(H1N1), both carrying the oseltamivir-resistant
mutation H275Y in the NA gene, were only partially
inhibited by 1 μM oseltamivir (Figure 5A and B, bottom
panels). A/Aichi/2/68(H3N2) was also not sensitive
to oseltamivir inhibition (Figure 5A and B, bottom
right graphs). Importantly, all viruses were inhibited
by high (17.5 μM) oseltamivir concentration (Figure 5B,
P<0.01), thus enabling us to effectively block NA ac-
tivity in order to study HA interactions with mucus
Figure 5 Sia cleavage preference and susceptibility to oseltamivir inhibition. (A) The cleaving preference of IAV NA was tested by
incubation of IAV with the fluorescent reporter, 4-methyl-umbelliferyl (4MU), linked to Neu5Ac, Neu5Gc, or 2-keto-3-deoxynononic acid (Kdn) as
substrate. All virus strains cleaved Neu5Ac and Neu5Gc but not Kdn. Background from spontaneous degradation of the 4MU-Sia compounds was
subtracted from the results. (B) Virus susceptibility to oseltamivir inhibition was tested by incubation of IAV with 2.5 nmol 4MU-Neu5Ac in the
presence of 0, 1 or 17.5 μM oseltamivir in triplicates. Certain strains were only partially inhibited by 1 μM oseltamivir, however, all of the strains
were inhibited by 17.5 μM oseltamivir. Substrate cleavage was quantified by measuring fluorescence from the released 4MU reporter compound.
*P<0.05, **P<0.01 (two-tailed T-Test, n=3).
Cohen et al. Virology Journal 2013, 10:321
Page 7 of 13
HSM directly inhibits viral neuraminidase
The ability of HSM and PSM to compete 4MU-Neu5Ac
for the virus NA activity was tested by incubating virus
with 4MU-Neu5Ac in the presence of mucus with 10
nmole Sia content (Figure 6). HSM but not PSM competi-
tively inhibits the cleavage of 4MU-Neu5Ac by A/Aichi/2/
68(H3N2) virus (Figure 6A). Similarly, HSM (4.7 nmol
Sia content) inhibited the cleavage of 4MU-Neu5Ac (0.1
nmol) by A/Denver/1/57(H1N1), A/Aichi/2/68(H3N2),
and A/SD/1/2009(SOIV). For all viruses PSM did not
inhibit cleavage of 4MU-Neu5Ac (Figure 6B). Since NA
affinity to 4MU-Neu5Ac compound is high , these
findings further confirm that HSM is effectively bound
by the enzymatic pocket of IAV NA.
Direct binding of IAV to HSM on magnetic beads array
IAV binding to sialylated mucus was tested incubating
virus with HSM and PSM conjugated to magnetic
beads. As control, we used magnetic beads conjugated
to a non-sialylated mucus-like polyacrylamide polymer.
A/PR/8/34(H1N1), A/SD/1/2009(SOIV) and A/Aichi/2/
68(H3N2) viruses (32-64 HAU) were incubated with the
beads for 1 h at 37°C. In order to avoid cleavage of sialy-
lated epitope and release of the virus, 16 μM oseltamivir
was added to the virus and to the wash buffer. Following
incubation, the beads were washed extensively to re-
move both unbound virus and oseltamivir. NA regains
normal activity once oseltamivir was removed (data not
shown). In order to quantify the bead-bound virus, each
sample was incubated with 10 nmol 4MU-Neu5Ac com-
pound for 30 min at 37°C, in the absence of oseltamivir.
The release of fluorescent 4MU compound directly corre-
lates with the number of virions in the sample. All three
tested strains bound to HSM, but only A/PR/8/34(H1N1)
bound to PSM (Figure 7). In addition virus binding to
magnetic beads conjugated to an array of sialylated poly-
acrylamide polymer standards was tested (Additional
file 4). The virus-binding pattern to the standard array was
in agreement with previous reports [33-36] (Additional
file 4). This confirms that the glycan array method pro-
duces reliable results. Thus the balance between HA
binding- and NA cleaving- of the sialylated mucus pro-
tective layer determines the ability of mucus to protect
underlying cells from infection.
We studied the interactions between IAV and host se-
creted mucins. Mucins are highly sialylated secretions
usually standing (or rather flowing) between the viruses
and their target cells on host epithelia. They are part of
a “chemical shield”, packed with defensive molecules
and innate immune cells , and also form a mechan-
ical clearance mechanism [8,38,39]. The small number
of existing studies of the human saliva inhibitory activity
on IAV infection identify several salivary molecules as
potential inhibitors including surfactants, secretory IgA,
histatins, defensins and MUC5B mucin [28,40]. In order
to focus on the sialylated mucin aspect of the respira-
tory tract, we enriched the mucin component of human
saliva and porcine submaxillary mucus samples by acid
precipitation. Since the Sia content of the mucus samples
was controlled throughout the experiments we were able
to provide direct experimental evidence for the mechan-
ism of mucus-protection during initial IAV infection.
We measured interactions of several different IAV
strains with mucins in vitro. IAV bind to Sias on mucins
(Figures 1, 2, and 7), and actively remove Sias from some
mucin targets in vitro (Figure 4). We further show that a
layer of HSM protects underlying cells from infection in
Sia-dose dependent manner (Figure 3) and that this pro-
tection is augmented when viral NA (sialidase) is inhib-
ited by oseltamivir in vitro (Figure 3A-B, gray bars). This
inhibition was dependent on the source of mucins.
Three of the IAV strains that were tested were not inhib-
ited by PSM (Figure 3A-B, and D) but all of the tested
strains were inhibited by HSM (Figure 3). Despite having a
Figure 6 HSM inhibits IAV cleavage of the 4MU-Neu5Ac reporter substrate. (A) Cleavage of 4MU-Neu5Ac reporter substrate by A/Aichi/2/68
(H3N2) was tested in the presence of HSM from two donors, PSM (10 nmol Sia), or PBS buffer. HSM from both donors inhibited the cleavage of
4MU-Ne5Ac. In contrast PSM did not inhibit cleavage of 4MU-Neu5Ac, similar to the buffer control. (B) Cleavage of 0.1 nmol 4MU-Neu5Ac by
three IAV strains was tested in the presence of HSM, PSM (4.7 pmol Sia), or PBS buffer in triplicates. All virus strains were inhibited by HSM but
not by PSM. Bars represent standard error, *P<0.05, **P<0.01 (two-tailed T-Test, n=3).
Cohen et al. Virology Journal 2013, 10:321
Page 8 of 13
similar Neu5Ac content (1,800 and 1,500 pmol Neu5Ac,
respectively), PSM (high) and HSM (low) do not have
the same inhibitory effect on IAV infection. HSM (low)
significantly reduced viral infection of three strains (A/
PR/8/34(H1N1), A/SD/17/2008(H1N1), and A/Aichi/2/
68(H3N2)), but PSM (high) only affected one of the
strains (A/SD/17/2008(H1N1)) and was less efficient
compared with HSM (low) (Figure 3C). Similarly, HSM
competitively inhibited sialidase activity of viral NA as
measured by 4MU-Sia cleavage assays, but PSM did not
(Figure 6). Inhibition of NA activity with 1 μM oselta-
mivir reduced the number of infected cells in HSM-
coated monolayers down to <10%, depending on the
strain (Figure 3A-B), thus exhibiting an additive inhibi-
tory effect. The NA of certain H1N1 strains, including
the pandemic A/California/04/2009(H1N1) contains a
functional secondary Sia-binding site, which can bind
oseltamivir . Indeed oseltamivir reduced the number
of cells infected with A/SD/2009(SOIV) by ~60% even
in the absence of mucus (Figure 3B); a similar trend was
observed for the other two H1N1 strains (Figure 3A and
C), but not for the H3N2 strain (Figure 3D). However,
an additive inhibitory effect was observed only for the
HSM-coated cells (Figure 3A-B, see Additional file 2 for
complete statistical analysis). Thus oseltamivir impairs
both the escape from sialylated mucin decoy and the
binding to target cells, by pandemic IAV.
Incidentally, early studies of human saliva have reported
the presence of Neu2en5Ac (N-Acetyl-2,3-dehydro-2-
deoxyneuraminic Acid) which is a very potent sialidase
inhibitor [41,42]. Modifications at the C-5 position of
Neu2en5Ac were the first improved in vitro NA inhibitors
. However, neither Neu2en5Ac nor the modified com-
pound has an in vivo inhibition activity against IAV,
possibly due to their rapid clearance . The presence of
Neu2enAc in natural mucins might indicate that hosts
also secrete their own NA-inhibitors. Alternatively, the
presence of Neu2enAc could be due to activity of siali-
dases in saliva as this molecule also represents an
intermediate product of sialidase activity.
Binding specificity of IAV strains is often studied using
glycan arrays . These are highly informative but lack
the 3D aspect of host cells as well as the overlaying
secretions. Mucins can be conceived as the “fluid glycan
arrays of nature” thus utilizing mucins for probing viral
functions could be immensely informative. Furthermore,
due to the constant clearance of airway mucus layer, at-
tenuating the rate of IAV penetration through the mucus
layer may be sufficient to prevent infection of the under-
lying epithelium. Considering the additive inhibitory ef-
fect of mucins and NA inhibition, it may be possible to
design NA-inhibiting drugs that will have minimal side
effects. Humans and other mammals express four NA
genes (NEU 1-4), which are present in the brain among
other tissues . It is conceivable that some of the re-
ported neuropsychiatric adverse effects of oseltamivir
and zanamavir [45,46] could be caused by cross-reaction
with endogenous NA in the brain. Our results warrant
further studies to better establish the function of viral
NA during in vivo infection and to establish ways by
which its role during initial infection could be perturbed.
Although most studies to date have focused on the po-
tential ability of NA inhibitors to prevent egress of viral
particles from infected cells, our study confirms that
these agents might also have a critical impact on viral in-
gress. Specifically we demonstrate that NA role during
initial infection includes cleavage of sialylated mucin de-
coys to allow virus penetration through the mucus layer.
Figure 7 Direct binding of IAV to HSM. Direct binding of IAV to bead-bound HSM and PSM was tested. Virus was incubated with the
mucus-coated beads in the presence of oseltamivir to inhibit NA activity. Following incubation, beads were washed to remove both
non-bound virus and oseltamivir. The bound virus was quantified by measuring NA activity using 4MU-Neu5Ac-reporter method. Average
of three independent experiments is shown. As control for non-specific binding, beads conjugated to non-sialylated polymer were used.
Black line indicates the background. All virus strains bound to HSM, but only one strain bound to PSM.
Cohen et al. Virology Journal 2013, 10:321
Page 9 of 13
Thus a better understanding of the NA and HA interac-
tions within the complex molecular “ecosystem” of host
mucins during initial infection also promises new avenues
for drug testing and development.
In this study, we show that mucins protect underlying
cells from IAV infection in a sialic acid-dose dependent
manner. We demonstrate direct binding to- and cleavage
of- sialylated human mucins by multiple IAV strains.
Our findings extend previous studies of inhibition by
oseltamivir , providing experimental evidence for the
specific molecular function of NA during initial infection.
We show that NA sialidase activity is required to free vi-
rions from sialylated host mucins decoy. Thus sialylated
host mucins have an important protective role against IAV
infection. Sia composition, presentation and density, are
critical for effective inhibition of the virus. Understanding
IAV interactions with the complex molecular “ecosystem”
of sialylated host mucins promises new avenues for drug
testing and development.
Viruses and cultured cells
Three influenza strains A/PR/8/34(H1N1), A/Denver/1/57
(H1N1) and A/Aichi/2/68(H3N2) were purchased from
ATCC. In addition, three clinical virus isolates A/SD/21/
2008(H1N1) and A/SD/17/2008(H1N1) and A/SD/1/2009
(SOIV) [47,48] were also used in this study. MDCK cells
were maintained in Dulbeco’s modified Eagle’s medium
(DMEM, Cellgro) supplemented with 10% fetal calf serum
(FCS). All viruses were propagated in MDCK cells that
were transferred to DMEM medium supplemented with
0.2% BSA fraction V (EMD), 25 mM HEPES buffer
(Gibco), 2 μg/ml TPCK-trypsin (Worthington Corpor-
ation), and 1% penicillin/streptomycin (“DMEM-TPCK”).
Antibodies and lectins
Anti-Influenza A nucleoprotein monoclonal antibody
(Anti-NP, MIA-NP-108) was purchased from eEnzyme,
TKH2 monoclonal antibody (HB-9654) was purchased
from ATCC, biotinylated Sambucus nigra lectin (bSNA)
was purchased from Vector laboratories. Biotinylated
donkey-anti-mouse IgG antibody and streptavidin conju-
gated horseradish peroxidase (SA-HRP) were purchased
from Jackson Immunoresearch.
Biotinylated probes and mucins
Biotinylated-polyacrylamide (PAA) glycan probes were
purchased from GlycoTech. Probes used in this study
were: Neu5Ac-PAA-biotin, Neu5Gc-PAA-biotin, Neu5Ac-
tin, 3’ Sialyllactose-PAA-biotin, 6’ Sialyllactose-PAA-biotin,
and Galβ1-3GalNAc-PAA-biotin. Human submaxillary
mucins (HSM) and porcine submaxillary mucins (PSM)
were biotinylated with EZ-link-NHS-PEG4-biotin (Thermo
Scientific) according to the manufacturer’s instructions.
Free biotin was removed by dialysis against PBS in a Slide-
A-Lyzer dialysis device (Thermo Scientific).
Salivary mucins were produced as previously described
with slight modifications . Briefly, human saliva was
collected from healthy human donors into tubes and
placed on ice, with the approval of the UCSD Human
Research Protections Program (protocol #080011). Saliva
samples were centrifuged 5 min at 10,000 g, and the pellet
was discarded. One percent penicillin/streptomycin was
added to the clear supernatant. Mucins were precipitated
by adjusting to pH 3.5 with 50 mM hydrochloric acid
(HCl), stirring over night at 4°C, and collecting at 800 g
for 10 min. The pellet was washed with miliQ water, re-
suspended in miliQ water, adjusted to pH 7.0 and boiled
for 10 min to inhibit protease and glycosidase activity.
Mucins were dialyzed against water in a 10,000 MWCO
Slide-A-Lyzer dialysis device (Thermo Scientific), and
stored in aliquots at -80°C. PSM was prepared following a
published protocol . The purity of the mucins was de-
termined by analysis of monosaccharide composition
using high performance anion exchange chromatography
with Pulsed Amperometric Detection (HPAEC-PAD).
In addition, amino acid content was determined by
Gas Chromatography Mass Spectrometry (GC-MS)
methods (Additional file 1) , both done at the UCSD
Glycotechnology Core Resource.
Sialic acid quantification by DMB-HPLC
Sia content was determined by 1,2-diamino-4,5-methyle-
nedioxybenzene dihydrochloride–high performance liquid
chromatography analysis (DMB-HPLC) according to a
published protocol . Samples were incubated for 1 h
with 0.1 M HCl at 80°C to release Sias, and filtered through
microcon-10kDa filtration device (Millipore). Free Sias
were incubated for 2.5 h at 50°C in the dark with 7 mM
DMB (Sigma), 0.75 M 2-mercapto-ethanol, 18 mM
Na-hydrosulfite in 1.4 M acetic acid. Sias were separated
on 250×4.6 mm Gemini C18 column (Phenomenex) with
7% MeOH, 8% Acetonitrile, 85% H2O solution at 0.9 ml/
min using the ELITE Lachrom HPLC system (Hitachi).
DMB-labeled Sias were detected at EX=373 EM=488.
Virus tissue-binding assay
Snap frozen human tissues were obtained as part of the
UCSD approved IRB protocol #101754. Tissues were
embedded in optimal cutting media (OCT) and cut into
5 micrometer-thick sections by Leica CM1800 cryomi-
crotome. Tissues were air-dried and fixed for 30 min in
10% buffered formalin, blocked with an Avidin/Biotin
Cohen et al. Virology Journal 2013, 10:321
Page 10 of 13
blocking kit according to the manufacturer’s instructions
(Vector Laboratories), followed by a 20 min incubation
with 0.3% H2O2in PBS, and a 10 min incubation with
1% bovine serum albumin in PBS (BSA/PBS). This
standard procedure does not affect glycan presentation
or virus binding to the tissue. Virus was concentrated
using a microcon-10 filtration system (Millipore); approxi-
mately 600 hemagglutinating Units (HAU) were mixed 1:1
with 1% BSA/PBS and incubated on the tissues for 1.5 h at
room temperature . The virus was fixed after rinsing
with 1:1 methanol/acetone for 15 min at -20°C. Tissue sec-
tions were then incubated with Anti-NP antibody (1:100)
for 1 hour at room temperature, followed by 30 min incu-
bation with biotinylated donkey-anti-mouse IgG antibody
(1:500), and a 30 min incubation with SA-HRP (1:500). As
a control for sialic acid specific binding, some tissue
sections were incubated with 0.25 mM Arthrobacter
ureafaciens sialidase in 50 mM sodium Acetate buffer
for 2 hours at 37°C, or with 2 mM ice-cold sodium period-
ate for 30 min at 4°C prior to formalin fixation. Tissues
were also stained with TKH2 antibody (1:50) over night at
4°C, followed by 30 min incubation with biotinylated-
donkey-anti-mouse-IgG antibody (1:500), and 30 min with
SA-HRP (1:500), or with biotinylated SNA (1:1000) 1 h at
room temperature, followed by 30 min incubation with
SA-HRP (1:500). Color was developed using Chromagen
AEC (SK 4200, Vector laboratories) and nuclei were coun-
terstained with Mayer Hematoxylin (Sigma-Aldrich). All
antibodies and lectins were diluted in 1% BSA/PBS.
Hematoxylin and Eosin (H&E), and Periodic Acid Schiff
(PAS, Electron Microscopy Sciences) staining was done as
previously described . Microscopy slides were scanned
with a NanoZoomer microscope (NanoZoomer 2.0 series,
Infection inhibition assay
MDCK cells in 16-well Lab-Tek chamber slides (Nunc,
Thermo Fisher Scientific, NY) were washed with PBS,
and layered with 60 μl HSM or PSM diluted in PBS
with Ca2+and Mg2+(Gibco) to 30, 64, or 240 pmol
Sia/μl, or with 60 μl PBS with Ca2+and Mg2+. Virus
was diluted in DMDM-TPCK media to 109TCID50/ml,
and aliquot into two vials, one of the vials contained 2 μM
oseltamivir. TCID50 of the virus was calculated according
to Spearman-Karber method. The cells were inoculated
with 60 μl virus for 1 h at 37°C, washed three times with
PBS, and fresh DMEM-TPCK media was added. Following
5.5 h incubation at 37°C, the cells were washed with PBS,
fixed for 20 min with 3% paraformaldehyde, permeabilized
for 4 min with 0.2% Triton X-100 (Sigma) in PBS, and
blocked with 1% BSA/PBS. Media chamber and gasket were
detached from the slides and the cells were incubated with
Anti-NP antibody (1:100) for 1 hour at room temperature,
followed by 30 min incubation with Alexa-488 conjugated
donkey-anti-mouse IgG antibody (1:500), Both antibodies
were diluted in 1% BSA/PBS. Nuclei were counterstained
with Hoechst stain (Life Technologies, NY). Samples were
analyzed with DeltaVision Deconvolution Microscope
equipped with Coolsnap HQ camera (Applied Precision,
WA). Twenty randomly selected images were taken at ×10
magnification from each sample, and the number of cells in
each image was determined using Volocity image analysis
software (PerkinElmer, MA). The experiment was repeated
three times. Data was analyzed by 3-way ANOVA, cor-
rected for multiple comparisons using Tukey’s HSD.
4MU-Sia cleavage assay
NA activity was measured using 2’-(4-methylumbelliferyl)-
α-D-N-acetylneuraminic acid (4MU-Neu5Ac)(Sigma-
Aldrich), 4MU-N-glycolyl-neuraminic acid (4MU-Neu5Gc)
(both gifts from Ken Kitajima, Nagoya University, Japan)
as a substrate as previously described . Briefly, 4MU-
Sia compounds were used as substrates in 33 mM 2-(N-
Morpholino)ethanesulphonic acid (MES, Sigma-Aldrich),
120 mM NaCl2, 4 mM CaCl2buffer adjusted to pH 6.5
(MES/CaCl2/NaCl2 buffer). In 96-well plates, 20 μl
4MU-Sia was added to achieve a final concentration of
300-10,000 pmol sia/well. Then 30 μl virus diluted in
MES/CaCl2/NaCl2buffer was added, and incubated for
1 h at 37°C in dark. For some wells oseltamivir was
added at a final concentration of 1 μM or 17.5 μM prior
to incubation. HSM and PSM inhibition of 4MU-Sia cleav-
age was done by adding mucus at 4,500-10,000 pmol sia/
well to wells containing 25-400 pmol 4MU-Neu5Ac prior
to addition of virus. The reaction was stopped by adding
150 μl of 25% ethanol, 0.1M glycine pH 10.7 and mea-
sured at excitation 365 nm and emission 450 nm in a
SpectraMax M3 spectrophotometer (Molecular Devices).
Magnetic beads sialoglycan array
Sera-Mag SpeedBeads Blocked Streptavidin (Thermo-Fisher
cat# 2115-2104-011150) magnetic beads were washed
in PBS, and incubated with biotinylated-HSM (bHSM),
biotinylated-PSM (bPSM) or biotinylated-polyacrylamide
(PAA) glycan probes in 125 mM PBS pH 7.4 for 1 h at
room temperature. The glycan-conjugated beads were
washed with PBS and incubated with virus diluted in PBS
containing 16 μM oseltamivir to 32-64 HAU for 1 h
at 37°C with rotation. Beads were washed three times
with oseltamivir-containing PBS, and once with PBS.
NA activity of bead-bound virus was quantified by
adding 100 nmol of 4MU-Neu5Ac and incubating for
30 min at 37°C. Cleavage of 4MU-Neu5Ac, quantified as
described above, is directly proportional to the number of
IAV bound to the beads.
Cohen et al. Virology Journal 2013, 10:321
Page 11 of 13
Mucus cleaving assay
Sera-Mag SpeedBeads Blocked Streptavidin (Thermo-Fisher)
magnetic beads were washed in PBS, and incubated
with bHSM, bPSM, or Galβ1-3GalNAc-PAA-biotin in
125 mM PBS pH 7.4 for 1 h at room temperature.
The glycan-conjugated beads were washed, resuspended
in 50 μl MES/CaCl2/NaCl2 buffer, and 50 μl virus
(600-2048 HAU) was added. As a control, 50 μl
DMEM-TPCK media was added to the beads. Beads
were incubated for 1.5 h at room temperature with
rotation, washed extensively with PBS, and fixed with
10% buffered formalin for 30 min. Beads were
washed, 100 μl 0.1 M HCl was added and Sias were
released from the beads by a 30 min incubation at
80°C. Sias were then quantified by DMB-HPLC.
Galβ1-3GalNAc-PAA-biotin conjugated beads were
used as control for non-specific signal.
The data sets supporting the results of this article are
included within the article and its additional files.
Additional file 1: Characterization of mucus samples. (A) Mucin
composition in the HSM preparation is comparable to that of the human
airway epithelium submucosal glands. (B) Amino acid composition was
analyzed by gas chromatography mass spectrometry, and (C)
monosaccharide composition was analyzed by high performance anion
exchange chromatography with Pulsed Amperometric Detection
(HPAEC-PAD). (D) The Sialic acid (Sia) content of PSM and HSM was
analyzed by DMB-HPLC. Sias in HSM are linked Neu5Acα2-6GalNAc
or Neu5Acα2-3Galβ1-3GalNAc , in PSM Sias are found as
Neu5Acα2-6GalNAc or Neu5Gcα2-6GalNAc . (E) Boiling of HSM samples
for 10 min inactivates bacterial sialidase activity, which is typically found in
Additional file 2: Complete statistical analysis for Figure 3. Results
of 3-way ANOVA analysis of the data presented in Figure 3, corrected for
multiple comparisons using Tukey’s HSD.
Additional file 3: Spontaneous degradation of 4MU-Sialic acid
compounds. All three fluorescent reporter compounds, 4-methyl-umbelliferyl
(4MU), linked to Neu5Ac, Neu5Gc, or 2-keto-3-deoxynononic acid (Kdn)
spontaneously degrade during 1 h incubation at 37°C. Notably 4MU-Neu5Gc
is the least stable compound. The fluorescent resulting from spontaneous
degradation of the reporter compounds was accounted as background and
was either subtracted from the results (Figure 5A) or presented as
background (Figure 5B).
Additional file 4: IAV binding to bead glycan array. Binding
specificity of IAV to sialylated glycoconjugates and mucus was tested on
bead-glycan array. Virus binding to the array was quantified by measuring
NA activity using 4MU-Neu5Ac-reporter method. Average of three
independent experiments, each done in triplicates, is shown. Black line
indicates the background. All virus strains bound to HSM. (A) A/PR/8/34
(H1N1) showed a preference for Siaα2-3Lactose over Siaα2-6Lactose, did
not bind to Neu5Gc containing structures, and showed weak binding to
PSM. (B) The pandemic strain A/SD/1/2009(SOIV) exhibited a broad
binding specificity, but did not bind PSM. (C) A/Aichi/2/68(H3N2)
showed preference for Sia in α2-6 linkages (Siaα2-6Lactose and Sialyl Tn),
had weak binding to Neu5Gc containing structures (Neu5Gc and
Neu5Gc-SialylTn), and did not bind to PSM.
The authors declare that they have no competing interest.
MC designed and carried out the experiments, analyzed the data and wrote
the manuscript. X-QZ isolated, characterized, subtyped the viruses and
analyzed data. HPS analyzed data. H-WC subtyped the viruses. NMV
interpreted the histology data. RTS designed the experiments and
interoperated the data. PG designed the experiments, interpreted the data
and wrote the manuscript. All authors read and approved the final
We thank Dr. Ken Kitajima (Nagoya University, Japan) for providing the 4MU-
N-glycolyl-neuraminic acid and 4MU- 2-keto-3-deoxynononic acid
compounds. Dr. Biswa Choudhury at the Glycotechnology Core Facility at
UCSD for amino acid and monosaccharide analysis. Dr. Stevan A. Springer
(UCSD) for helping with the statistical analysis. Dr. Eillen Tecle (UCSD) for
helpful comments on the manuscript. Images were acquired at the
Neuroscience Microscopy Shared Facility, UCSD. Human tissues were
obtained as part of a UCSD approved IRB protocol. This work was supported
by the University of California Laboratory Fees Research Program Award
(118645), a Cooperative Agreement from the National Institute of Allergy and
Infectious Diseases (1U01AI074521), a grant from the National Institute of
Neurological Disorders and Stroke (P30 NS047101), and UCSD Cancer Center
Specialized Support grant (P30 CA23100).
1Department of Cellular and Molecular Medicine, University of California San
Diego, 9500 Gilman Dr, La Jolla 92093, California, USA.2Division of Infectious
Disease, University of California San Diego, 9500 Gilman Dr, La Jolla 92093,
California, USA.3Present address: School of Veterinary Medicine, National
Taiwan University, 1 Sec. 4 Roosevelt Rd, Taipei 10617, Taiwan.
Received: 17 September 2013 Accepted: 14 October 2013
Published: 22 November 2013
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Cite this article as: Cohen et al.: Influenza A penetrates host mucus by
cleaving sialic acids with neuraminidase. Virology Journal 2013 10:321.
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