Vol.2, No.9, 1005-1014 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.29123
Highly conserved domains in hemagglutinin of influenza
viruses characterizing dual receptor binding
Wei Hu
Department of Computer Science, Houghton College, Houghton, USA;
Received 4 June 2010; revised 15 July 2010; accepted 18 July 2010.
ABSTRACT
The hemagglutinin (HA) of influenza viruses in-
itiates virus infection by binding receptors on
host cells. Human influenza viruses preferenti-
ally bind to receptors with α2,6 linkages to gala-
ctose, avian viruses prefer receptors with α2,3
linkages to galactose, and swine viruses favor
both types of receptors. The pandemic H1N1
2009 remains a global health concern in 2010.
The novel 2009 H1N1 influenza virus has its ge-
netic components from avian, human, and sw-
ine viruses. Its pandemic nature is characterized
clearly by its dual binding to the α2,3 as well as
α2,6 receptors, because the seasonal human
H1N1 virus only binds to the α2,6 receptor. In pr-
evious studies, the informational spectrum me-
thod (ISM), a bioinformatics method, was appli-
ed to uncover highly conserved regions in the
HA protein associated with the primary receptor
binding preference in various subtypes. In the
present study, we extended the previous work
by discovering multiple domains in HA associa-
ted with the secondary receptor binding prefer-
ence in various subtypes, thus characterizing
the distinct dual binding nature of these viruses.
The domains discovered in the HA proteins
were mapped to the 3D homology model of HA,
which could be utilized as therapeutic and diag-
nostic targets for the prevention and treatment
of influenza infection.
Keywords: Binding Specificity; Discrete Fourier
Transform; Electron-Ion Interaction Potential;
Hemagglutinin; Influenza; Informational Spectrum
Method
1. INTRODUCTION
Influenza A viruses have two surface proteins, hemagg-
lutinin (HA) and neuraminidase (NA). HA is a hom-
otrimer, in which each monomer comprises two subdo-
mains, HA1 and HA2. HA1 initializes the contact with
the cell membrane and HA2 mediates membrane fusion.
In general, human influenza and avian viruses preferen-
tially bind to the α2,3 sialylated and α2,6 sialylated gly-
can receptors, respectively [1-3]. Pigs have receptors for
both human and avian influenza viruses on their tracheal
cells, thus they can serve as a mixing vessel to re-assort
genes from different species to make new influenza vir-
uses. The 2009 H1N1 pandemic was caused by a novel
swine-origin influenza A virus.
Using sequence analysis and homology modeling [4],
the HA protein of 2009 H1N1 was found to have the sig-
nature amino acid Asp190 and Asp225 known to confer
binding affinity to α2,6 sialylated glycan receptors. The
mutation Glu190Asp between avian and human H1 HA
normally would lead to the loss of a critical contact with
α2,3 glycans, which, however, was compensated by the
presence of Lys145 in HA of 2009 H1N1. There were
four loops in 2009 H1N1 HA, 130 loop, 140 loop, 150
loop, and 220 loop, each containing one Lys, to form a
positively charged ‘lysine fence’ at the base of the bind-
ing site to support optimal contacts with the α2,6 and
α2,3 receptors. Based on this structural analysis, it was
predicted that the HA protein of 2009 H1N1 virus can
bind to the α2,6 as well as α2,3 sialylated glycan recap-
tors, which was subsequently verified by the carbohyd-
rate microarray analysis in [5].
There were several other efforts in expanding the kno-
wledge on 2009 H1N1. One study [6] investigated the th-
ree aspects of NA: the mutations and co-mutations, the
stalk motifs, and the phylogenetic analysis. The potential
mutations and strongly co-mutated positions of NA were
found. A special NA stalk motif of high virulence, which
was dominant in the past H5N1 strains, was discovered
in H1N1 in 2009 for the first time. Another study [7]
focused on HA and the interaction between HA and NA.
The mutations of HA in 2009 H1N1 were found and ma-
pped to the 3D homology model of H1, and the muta-

W. Hu / Natural Science 2 (2010) 1005-1014
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tions on the five epitope regions on H1 were identified.
The distinct response patterns of HA to the changes of
NA stalk motifs were discovered, illustrating the functio-
nal dependence between HA and NA. With help from
the results of the first study [6], two comutation netw-
orks were uncovered, one in HA and one in NA, where
each mutation in one network co-mutates with the mu-
tations in the other network across the two proteins HA
and NA. These two networks residing in HA and NA
separately might provide a functional linkage between
the mutations that could change the drug binding sites in
NA and those that could affect the host immune respon-
se or vaccine efficacy in HA.
The genes of 2009 H1N1 were derived from avian,
human, and swine viruses. Identification of host shift ma-
rkers of this novel virus remains an urgent and important
research topic. However, sequence survey of 2009 H1N1
suggested the absence of the well-known host switch ma-
rkers. A new procedure was designed to locate a collec-
tion of novel host markers in ten proteins/genes of 2009
H1N1 [8,9], which included, in addition to the SR polym-
orphism found in [10], a set of markers in PB2 that mi-
ght play compensatory roles in the efficient replication
and transmission of this new virus. These novel markers
of 2009 H1N1 offered new and ample opportunities for
further investigation of their biological functions in host
adaptation to humans experimentally.
The informational spectrum method (ISM) [11] is a
bioinformatics technique to study the biological functi-
ons of proteins with their physicochemical properties,
which first translates a protein sequence into a numerical
sequence based on each amino acid’s electron-ion inter-
action potential (EIIP) and then the discrete Fourier tra-
nsformation (DFT) is applied to it to create an inform-
ational spectrum. It is believed that the protein functions
including the protein-protein interaction are encoded in
the peaks of the informational spectrum.
In [12,13] it was found that the CIS of HA1 of influe-
nza strains have the primary characteristic dominant pea-
ks at different IS frequencies as presented in Table 1. In
this study, F(0.295) will be referred to as pandemic hu-
man H1N1 receptor interaction frequency, F(0.055) as
swine receptor interaction frequency, F(0.076) as avian
receptor interaction frequency, and F(0.236) as seasonal
human H1N1 receptor interaction frequency. Some infl-
uenza strains display dual binding preference, one prim-
ary and one secondary, as demonstrated in [12,13].
In [12,13] the ISM was applied to investigate the inte-
raction between HA protein and its receptors, which sho-
wed that HA1encodes highly conserved domains essenti-
al for receptor binding affinity. Their analysis also found
the following receptor recognition domains in HA prote-
ins from H1N1, H3N2, H5N1, and H7N7 (Table 2).
One region in HA1 of various strains was found to be
connected with the primary receptor binding preference
in [12] (Table 2). In [14], multiple such regions in HA1
responsible for the primary receptor binding preference
in different subtypes were discovered (Table 3). In a stu-
dy of receptor binding specificity of influenza viruses
[14], the ISM was applied to find the mutations in HA1
and to elucidate the contribution to receptor binding
switch from each mutation quantitatively. Two clusters
of mutations in HA1 of 2009 H1N1 were uncovered,
where the first was at residues 152-170 and the second at
residues 257-278. The first cluster of mutations was
contained in a pandemic human H1N1 receptor recogni-
tion domain (150:174) with the primary characteristic IS
frequency at F(0.295) found in [15] (Table 3). Prompted
by this finding, we searched for a similar domain in the
vicinity of the second cluster, and found one such dom-
ain (246:286) associated with swine binding preference
at the secondary characteristic IS frequency F(0.055) [15]
(Table 3). Our aim in this study was to extend these
results by identifying multiple domains in HA1 of influ-
enza viruses associated with the secondary receptor
binding preference, thus providing the complete infor-
mation about the dual binding of the HA proteins. These
conserved domains in HA1 might be used to develop
targets for new drugs and infection control.
2. MATERIALS AND METHODS
2.1. Sequence Data
All HA sequences were retrieved from the Flu Resource
Table 1. Primary characteristic IS frequencies of HA proteins
in 2009 H1N1, swine H1N1/H1N2, avian H5N1, and seasonal
human H1N1.
Subtype 2009
H1N1
Swine
H1N2/H1N1
Avian
H5N1
Seasonal human
H1N1
FrequencyF(0.295)F(0.055) F(0.076) F(0.236)
Table 2. The receptor recognition domains of HA proteins in
H1N1, H3N2, H5N1, and H7N7 influenza viruses.
Strain Frequency Residues
A/California/04/2009 (H1N1) F(0.295) 284-326
A/Hong Kong/213/03 (H5N1) F(0.076) 42-75
A/New Caledonia/20/99 (H1N1) F(0.236) 262-295
A/New York/383/2004 (H3N2) F(0.363) 57-90
A/equine/Prague/56 (H7N7) F(0.285) 28-61
A/Egypt/0636-NAMRU3/2007(H5N1) F(0.236) 99-132
A/South Carolina/1/18 (H1N1)) F(0.258) 87-120

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Table 3. The receptor recognition domains of HA proteins in 2009 H1N1, swine H1N2, and avian H5N1
influenza viruses [14,15].
Subtype Frequency Residues Consensus HA1 Sequence
2009 H1N1 F(0.295) 106-130 SSVSSFERFEIFPKTSSWPNHDSNK
2009 H1N1 F(0.295) 150-174 WLVKKGNSYPKLSKSYINDKGKEVLV
2009 H1N1 F(0.295) 191-221 LYQNADAYVFVGSSRYSKKFKPEIAIRPKVR
Swine H1N2 F(0.055) 1-29 DTLCIGYHANNSTDTVDTVLEKNVTVTHS
Avian H5N1 F(0.076) 46-65 GVKPLILRDCSVAGWLLGNP
Avian H5N1 F(0.076) 136-153 PYQGRSSFFRNVVWLIKK
Avian H5N1 F(0.076) 269-286 LEYGNCNTKCQTPMGAIN
(http://www.ncbi /nlm.nih.giv /geno mes/FLU/FLU.html)
of the National Center for Biotechnology Information
(NCBI) on Nov. 20, 2009. Only the full length and un-
ique sequences were selected. There were 450 HA sequ-
ences of human 2009 H1N1, 1228 HA sequences of av-
ian H5N1 from 1959 to 2009, and 83 HA sequences of
swine H1N2 from 1980 to 2009. All the sequences used
in the study were aligned with MAFFT [16].
2.2. Important Sites in HA
Although there is a great variation due to high selection
pressure in the HA1 sequences of various flu subtypes,
the active site of HA1 is well conserved, which is loca-
ted in a cleft composed of the residues 91, 150, 152, 180,
187, 191, and 192 [17]. The three amino acids at positi-
ons 187, 191 and 192 are a part of the 190 helix. The ac-
tive site cleft of HA is formed by its right edge (131_
GVTAA) and left edge (221_RGQAGR) (H1 number-
ing), which are also commonly referred to as the 130 loop
and 220 loop, respectively [18].
2.3. Informational Spectrum Method
The informational spectrum method is a bioinformatics
technique that can be utilized to analyze protein sequen-
ces. Prior to this analysis, the protein sequences have to
be translated into numerical sequences. One such appro-
ach is to assign each amino acid to its electron-ion inter-
action potential (EIIP), which represents the average en-
ergy of the valence electrons in the amino acid (Table 4).
Table 4. The electron-ion interaction potential (EIIP) of amino
acids used to encode amino acids.
Amino acid EIIP Amino acid EIIP
L 0.0000 Y 0.0516
I 0.0000 W 0.0548
N 0.0036 Q 0.0761
G 0.0050 M 0.0823
E 0.0057 S 0.0829
V 0.0058 C 0.0829
P 0.0198 T 0.0941
H 0.0242 F 0.0946
K 0.0371 R 0.0959
A 0.0373 D 0.1263
The application of EIIP to protein function analysis assu-
mes that the strength of the electromagnetic field surrou-
nding the protein is indicative of its biological function.
This method was successful in revealing various protein
properties [11].
The numerical sequence x(m) of a protein sequence is
transformed into the frequency domain using DFT. The
DFT coefficients X(n) are defined as
2
()( )1,2,2
jnm
N
X
nxme n N
 




where N is the length of sequence x(m).
The energy density spectrum is defined as
 
2,1,2,,2SnXnX nXnnN


The informational spectrum (IS) of a sequence x(m)
comprises the frequencies and the amplitudes of its DFT.
Peak frequencies of IS of a protein sequence reflect its
biological or biochemical functions. To determine the sa-
me biological or biochemical functions of a group of pro-
tein sequences, a consensus informational spectrum (CIS)
can be used, which is defined as the product of energy de-
nsity spectrum S(n) of each sequence in the group. A me-
asure of similarity for each peak is a signal-to-noise ratio
(S/N), which is defined as a ratio of signal density to the
mean value of the whole spectrum [11].
2.4. Consensus HA1 Sequences
We employed MAFFT to align the three consensus HA1
sequences of 2009 H1N1, avian H5N1, and swine H1N2
(Figure 1). Each consensus sequence was then used in
the ISM analysis to find the highly conserved domains in
HA1 of different influenza subtypes.
3. RESULTS
As demonstrated in [12-15], the HA1 sequences in vari-
ous influenza subtypes had a distinct inclination to inte-
ract with a specific primary receptor as well as a specific
secondary receptor, and there were regions in HA1 enco-
ding highly conserved information associated with the

W. Hu / Natural Science 2 (2010) 1005-1014
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Figure 1. Multiple sequence alignment of three consensus HA1
sequences of 2009 H1N1, swine H1N2, and avian H5N1 (pre-
sented in this order as well). The binding sites in HA are col-
ored in red, the left and right edges of the binding cleft in blue.
primary binding preference. The main task of the present
study is to explore the other parts of the HA1 sequences
to find domains associated with the secondary binding
preference. The entropy of HA1 of 2009 H1N1, swine
H1N2, and avian H5N1 was calculated in [14], illustrat-
ing the most conserved positions in the HA1 sequences
of each subtype. The ISM bioinformatics technique was
applied to the three consensus HA1 sequences as prese-
nted in Figure 1 to uncover the conserved domains in
HA1, which might affect the secondary receptor speci-
ficity in each subtype. In contrast, the ISM analysis in
[12,13] was applied to a particular selected strain in a
subtype such as A/California/04/2009 in 2009 H1N1 to
find a conserved region. Overall, the conserved domains
discovered by our approach using a consensus sequence
had more coverage to different strains in a subtype than
the single strain approach used in [12,13].
3.1. Conserved Domains in HA1 of 2009
H1N1
Using the ISM, we discovered seven conserved domains
in HA1 of 2009 H1N1 responsible for swine binding pr-
eference, which were located at residues 5-23, 63-81,
142-158, 178-195, 201-216, 246-269, and 277-287, re-
spectively (Figure 2). The ISs of these domains and the
consensus HA1 sequence in 2009 H1N1 were plotted in
Figure 3. The IS of the consensus HA1 sequence in 2009
H1N1displayed a primary dominant peak of frequency
F(0.295) and a secondary prominent peak of frequency
F(0.055), demonstrating its dual binding preference. The
domain 142:158 contained two binding sites 150 and 152
Figure 2. The two plots show in 3D structure the seven highly
conserved domains related to swine binding characteristic fou-
nd in HA of 2009 H1N1. Domain 5:23 is colored in red, dom-
ain 63:81 in blue, domain 142:158 in green, domain 178:195 in
orange, domain 201:216 in yellow, domain 246:269 in pink,
and domain 277:287 in gray (PDB code: 1RU7).
and was near the right edge of the active site. The dom-
ain 178:195 included four binding sites 180, 187, 191, and
192. The domains 201:216 and 246:269 were close to
the left edge of the active site, illustrating their important
roles in receptor binding preference. As seen from the
entropy distribution of HA1 of 2009 H1N1 in [14], these
seven domains were highly conserved. In [12], a similar
domain with human receptor binding characteristic was
found in the C-terminus of the HA protein consisting of
residues 284-326. Multiple such domains were identified
in [14], and one domain with swine receptor binding
characteristic was found in [15] (Table 1).
3.2. Conserved Domains in HA1 of Swine
H1N2
In [12], the sequences of swine H1N1 and H1N2 were
combined into a single dataset for analysis. Here the
swine H1N2 was treated as a single dataset. The con-
served domains found in this subtype were separated in
two categories, i.e., one had swine binding preference
(primary) and one had human binging preference (sec-
ondary). The four domains with swine binding were at
residues 63-81, 178-189, 241-271, and 277-287, respec-
tively (Figure 4), and the four domains with human
binding were at residues 104-129, 189-213, 240-265,
and 271-301, respectively (Figure 5). The ISs of these
sequence in swine H1N2 revealed two dominant peaks at

W. Hu / Natural Science 2 (2010) 1005-1014
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0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
6
5
4
3
2
1
0
Fre
q
uenc
y
Amplitude
IS of consensus HA1 sequence of 2009 H1N1
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequenc
y
Am
p
litude
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (5:23) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (63:81) associated with
wine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (142:158) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (178:195) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (201:216) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (246:269) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
6
5
4
3
2
1
0
IS of a highly conserved domain (277:287) associated with
swine receptor interaction
Figure 3. The eight plots show the informational spectrum of consensus HA1 sequence in 2009 H1N1 and that of the seven con-
served domains in HA1 of 2009 H1N1 connected with swine receptor binding characteristic (F(0.055)), respectively.
frequencies of F(0.055) (primary) and F(0.295) (second-
dary). The entropy analysis in [13] suggested that these
domains and the consensus HA1 sequence in swine H1-
N2 were displayed in Figure 6. The IS of the consensus
HA1 domains were well conserved. One such domain in
HA1 of swine H1N2 was located in the N-terminus of
the protein at residues 1-29 [13] (Table 3).
3.3. Conserved Domains in HA1 of Avian
H5N1
We found five conserved domains in HA1 of avian H5-
N1 relevant to human binding affinity, located at resid-
ues 9-39, 97-115, 123-139, 144-167, and 199-227, re-
spectively (Figure 7). The ISs of these five domains and

W. Hu / Natural Science 2 (2010) 1005-1014
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Figure 4. The two plots show in 3D structure the four
highly conserved domains of swine binding character-
istic found in HA of swine H1N2. Domain 63:81 is co-
lored in red, domain 178:189 in blue, domain 241:271
in green, and domain 277:287 in orange.
Figure 5. The two plots show in 3D structure the four
highly conserved domains of human binding characte-
ristic found in HA of swine H1N2. Domain 104:129 is co-
lored in red, domain 189:213 in blue, domain 240:265
in orange, and domain 271:301 in yellow.
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
7
6
5
4
3
2
1
0
Fre
q
uenc
y
Amplitude
IS of consensus HA1 sequence of swine H1N2
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Amplitude
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (63:81) associated with swine
receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequenc
y
Am
p
litude
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (178:189) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (241:271) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequ e nc
y
Am
p
litude
6
5
4
3
2
1
0
IS of a highly conserved domain (277:287) associated with
swine receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequenc
y
Am
p
litude
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (104:129) associated with
pandemic human H1N1 receptor interaction

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0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
9
8
7
6
5
4
3
2
1
0
IS of a highly conserved domain (189:213) associated with
pandemic human H1N1 receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
7
6
5
4
3
2
1
0
IS of a highly conserved domain (271:301) associated with
pandemic human H1N1 receptor interaction
IS of a highly conserved domain (240:265) associated with pandemic human H1N1 receptor interaction
Freq u enc y
Am
p
litude
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Figure 6. The nine plots show the informational spectrum of consensus HA1 sequence of swine H1N2, and that of seven conserved
domains in swine H1N2 associated with human and swine binding (F(0.295) and F(0.055)), respectively.
Figure 7. The two plots show in 3D structure the five highly
conserved domains found in HA of avian H5N1 related to hu-
man binding. Domain 9:39 is colored in red, domain 97:115 in
blue, domain 123:139 in green, domain 144:167 in orange, and
domain 199:227 in yellow (PDB code: 2IBX).
the consensus HA1 sequence in avian H5N1 were illus-
trated in Figure 8. The IS of the consensus HA1 seque-
nce in avian H5N1 demonstrated two dominant peaks of
frequencies F(0.076) (primary) and F(0.236) (secondary).
The entropy analysis in [14] implied that the five doma-
ins were well conserved, and the HA1 sequences of av-
ian H5N1 were quite stable, in contrast to those of 2009
H1N1 and swine H1N2. In [13], a similar domain with
avian binding was found in the N-terminus of the HA pr-
otein comprising residues 42-75. In [14], multiple such
domains with avian binding were uncovered (Table 3).
4. CONCLUSIONS
Identifying the conserved characteristics of influenza vi-
ruses relevant to receptor binding preference is of great
importance in flu research. The informational and struct-
ural properties of HA1 of influenza viruses associated
with the primary receptor affinity were investigated in
[12-15]. To extend the previous results, we sought to un-
cover multiple domains in HA1 associated with the seco-
ndary binding preference, thus to expand our repertoire
of these key regions in HA1 of influenza viruses.
The main findings of this study were presented in Ta-
ble 5, which showed the locations, the characteristic IS
frequencies, and the consensus sequences of the seven
highly conserved domains in HA1 discovered in differ-
ent subtypes. Combined with the results in Table 3 and
those in [12,13], these findings provided the complete

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Fre
q
uenc
y
Amplitude
IS of consensus HA1 sequence of avian H5N1
Frequenc
y
Am
p
litude
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (9:39) associated with
seasonal human H1N1 receptor interaction
0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
6
5
4
3
2
1
0
IS of a highly conserved domain (97:115) associated with
seasonal human H1N1 receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (123:139) associated with
seasonal human H1N1 receptor interaction
0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
6
5
4
3
2
1
0
IS of a highly conserved domain (97:115) associated with
seasonal human H1N1 receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Frequency
Am
p
litude
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
IS of a highly conserved domain (199:227) associated with
seasonal human H1N1 receptor interaction
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
IS of a highly conserved domain (240:265) associated with pandemic human H1N1 receptor interaction
Frequenc
y
Am
p
litude
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0 0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Figure 8. The seven plots show the informational spectrum of consensus HA1 sequence in avian H5N1 and the informational spec-
trum of the six conserved domains in avian H5N1 associated with human binding (F(0.236)), respectively.
information about the conserved domains in HA1 that
might modulate the dual receptor binding preference.
The domains in Table 5 are shorter than those discov-
ered in [12,13], implying that they were more easily co-
nserved by the HA sequences than their longer counterp-
arts. Furthermore, the identified multiple domains in HA1
related to dual receptor binding preference could provide
ample options to design new therapeutic targets for drug
development. Finally, because a consensus sequence of
each subtype was employed to find these multiple dom-
ains in HA1, they are more representative of the strains
in a given subtype than those obtained in [12,13].

W. Hu / Natural Science 2 (2010) 1005-1014
101
1013
Table 5. The receptor recognition domains in HA protein in 2009 H1N1, avian H5N1, and swine H1N2
influenza viruses.
Subtype Frequency Residues Consensus HA1 Sequence
2009 H1N1 F(0.055) 5-23 IGYHANNSTDTVDTVLEKN
2009 H1N1 F(0.055) 63-81 GNPECESLSTASSWSYIVE
2009 H1N1 F(0.055) 142-158 KSFYKNLIWLVKKGNSY
2009 H1N1 F(0.055) 178-195 GIHHPSTSADQQSLYQNA
2009 H1N1 F(0.055) 201-216 VGSSRYSKKFKPEIAI
2009 H1N1 F(0.055) 246-269 GNLVVPRYAFAMERNAGSGIIISD
2009 H1N1 F(0.055) 277-287 TTCQTPKGAIN
Swine H1N2 F(0.295) 104-129 QLSSVSSFERFEIFPKESSWPNHDTN
Swine H1N2 F(0.295) 189-213 QSLYQNADAYVFVGSSHYSKKFTPE
Swine H1N2 F(0.295) 240-265 ITFEATGNLVVPRYAFALKRGSGSGI
Swine H1N2 F(0.295) 271-301 SVHDCNTTCQTPKGAINTSLPFQNIHPVTIG
Swine H1N2 F(0.055) 63-81 GNPECESLFTASSWSYIVE
Swine H1N2 F(0.055) 178-189 GIHHPSTSADQQ
Swine H1N2 F(0.055) 241-271 TFEATGNLVVPRYAFALKRGSGSGIIISDTS
Swine H1N2 F(0.055) 277-287 TTCQTPKGAIN
Avian H5N1 F(0.236) 9-39 ANNSTEQVDTIMEKNVTVTHAQDILEKTHNG
Avian H5N1 F(0.236) 97-115 DYEELKHLLSRINHFEKIQ
Avian H5N1 F(0.236) 123-139 SDHEASSGVSSACPYQG
Avian H5N1 F(0.236) 144-167 FRNVVWLIKKNNAYPTIKRSYNNT
Avian H5N1 F(0.236) 199-227 SVGTSTLNQRLVPKIATRSKVNGQSGRME
It was suggested in [12,13] that the 2009 H1N1 strains
will continue to mutate in their HA gene, which could
further favor the human interaction pattern by increasing
the amplitude at frequency F(0.295) and at the same time
decreasing that at frequency F(0.055). Identification of
multiple domains in HA of influenza viruses related to
dual receptor binding preference provided relevant info-
rmation for the development of potential targets for new
drugs and treatment of influenza infection
5. ACKNOWLEDGEMENTS
We thank Houghton College for its financial support.
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