﻿Acquisition of Human-Type Receptor Binding Specificity
by New H5N1 Influenza Virus Sublineages during Their
Emergence in Birds in Egypt
Yohei Watanabe1
*, Madiha S. Ibrahim1,2¤
, Hany F. Ellakany3
, Norihito Kawashita4,5
, Rika Mizuike1
,
Hiroaki Hiramatsu6
, Nogluk Sriwilaijaroen6,7
, Tatsuya Takagi4,5
, Yasuo Suzuki6
, Kazuyoshi Ikuta1
*
1 Department of Virology, Research Institute for Microbial Diseases (BIKEN), Osaka University, Osaka, Japan, 2 Department of Microbiology, Faculty of Veterinary Medicine,
Alexandria University, Damanhour Branch, Egypt, 3 Department of Poultry Diseases and Hygiene, Faculty of Veterinary Medicine, Alexandria University, Edfina Branch,
Egypt, 4 Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, 5 Genome Information Research Center, Research Institute for Microbial Diseases,
Osaka University, Osaka, Japan, 6 Health Scientific Hills, College of Life and Health Sciences, Chubu University, Aichi, Japan, 7 Faculty of Medicine, Thammasat University
(Rangsit Campus), PathumThani, Thailand
Abstract
Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60%
mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1
virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A
change in receptor binding affinity of the viral hemagglutinin (HA) from a2,3- to a2,6-linked sialic acid (SA) is thought to be
necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated
between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally
into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics,
we found that these H5 sublineages have acquired an enhanced binding affinity for a2,6 SA in combination with residual
affinity for a2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1
viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased
attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with
enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to a2,6 SA, emerged
during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence
of new H5 sublineages with a2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in
Egypt, and provided data for understanding the virus's pandemic potential.
Citation: Watanabe Y, Ibrahim MS, Ellakany HF, Kawashita N, Mizuike R, et al. (2011) Acquisition of Human-Type Receptor Binding Specificity by New H5N1
Influenza Virus Sublineages during Their Emergence in Birds in Egypt. PLoS Pathog 7(5): e1002068. doi:10.1371/journal.ppat.1002068
Editor: Ron A. M. Fouchier, Erasmus Medical Center, Netherlands
Received December 22, 2010; Accepted March 30, 2011; Published May 26, 2011
Copyright: ß 2011 Watanabe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Grant-in Aid for Scientific Research (20406025) from the Ministry of Education, Culture, Sports, Science and Technology
(http://www.mext.go.jp/english/), Japan (Scientific Research (B), Overseas Academic Research) and Global COE Program from the Japan Society for the Promotion
of Science (http://www.jsps.go.jp/english/), Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: nabe@biken.osaka-u.ac.jp (YW); Ikuta@biken.osaka-u.ac.jp (KI)
¤ Current address: Department of Microbiology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt
Introduction
Since the emergence of highly pathogenic avian influenza virus
subtype H5N1 (HPAI H5N1) in 1996, outbreaks have continued
in a variety of domestic and wild birds as well as sporadic
transmission to humans [1]. Over time, H5N1 viruses have
diversified and are currently grouped into clades 0 to 9 according
to the unified nomenclature system [2]. Since 2006, clade 2.2,
which originated from a large outbreak in wild bird populations at
Qinghai Lake in western China [3,4], has spread rapidly over
central Asia, Europe, the Middle East, and Africa [5,6]. Clade 2.2
has further diversified forming the third-order clade 2.2.1 and
three phylogenetically distinct sublineages (I, II and III) within
clade 2.2 [7,8].
Although the current H1N1 pandemic [9] may have diverted
attention from the continuing worldwide circulation of H5N1
virus, the pandemic threat of H5N1 is still alarming. The
cumulative number of confirmed human cases of H5N1 infection
reported to the World Health Organization (WHO) to date is 504
with a 60% mortality [10]. According to the World Organization
for Animal Health, HPAI H5N1 has become endemic in some
areas where human cases constitute more than 80% of the total
[10], indicating bird-human H5N1 virus transmission; e.g., China,
Indonesia, Viet Nam and Egypt [11].
Since 2006, H5N1 viruses have spread across countries in
western, eastern, and northern Africa, where viruses belonging to
clade 2.2.1 and three sublineages (I, II and III) of clade 2.2 have
been detected [7,8]. As of October 2010, WHO has reported 114
laboratory-confirmed human cases on the African continent [10].
Egypt has experienced a relatively large number of human
infections with 112 confirmed cases reported since 2006, when
H5N1 was first identified in Egypt. In particular, the cumulative
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number since 2009 is notable: 61 confirmed cases in Egypt. The
worldwide number since is also 112 cases. This indicates that the
recent human H5N1 cases in Egypt are more than 50% of the
total worldwide. The other 2 human cases of H5N1 virus infection
in Africa were reported from Nigeria and Djibouti. The reason(s)
for such a high number of human H5N1 cases in Egypt has not
been elucidated.
Influenza viruses target glycosylated oligosaccharides that
terminate in a sialic acid (SA) residue [12­14]. These residues
are bound to glycans through an a2,3, a2,6, a2,8 or a2,9 linkage
by sialyltransferases that are expressed in a tissue- and species-
specific manner [15­17]. For example, human upper airway
epithelia express mostly a2,6-linked SA (a2,6 SA) [18], whereas
duck intestinal epithelia express mainly a2,3-linked SA (a2,3 SA)
[19]. Efficient human-human transmission is necessary for
influenza A virus to become pandemic. Although the determinants
of efficient human-human transmission are not fully understood, it
is believed that a change of receptor specificity from a2,3 SA, to
which avian influenza A viruses preferentially bind, to a2,6 SA, to
which human influenza viruses preferentially bind, is essential
[12,20,21]. Although H5N1 viruses still lack the ability for efficient
human-human transmission, the current prevalence of H5N1
might allow the virus to acquire mutations enabling a2,6 SA
recognition. Thus, it is important to monitor the receptor binding
affinity of H5N1 viruses in endemic areas and evaluate molecular
mechanisms that might promote their pandemic potential.
In this study, we carried out a phylogenetic analysis of avian and
human H5N1 viruses circulating in Egypt. The resulting virus
phylogenetic tree indicated emergence of new H5 sublineages with
each sublineage containing only or mostly human isolates, leading
us to hypothesize that the HAs of these viruses might have acquired
amino acid change(s) enabling a2,6 SA binding and resulting in the
large number of human H5N1 cases in Egypt. Therefore, in this
study we examined the receptor binding affinity of H5N1 viruses
isolated in Egypt using sialylglycopolymers and human respiratory
tract tissues, and assessed the effect of the amino acid changes in the
HAs on viral replication in human airway epithelia in vitro and
virulence in mice in vivo. We show here that these H5N1 viruses,
during their spread in local bird populations, acquired mutations in
their HAs that produced a2,6 SA binding affinity, providing a
model for influenza virus phylogeny.
Results
Phylogeny of H5N1 viruses circulating in Egypt
We studied the evolution of H5N1 influenza viruses in Egypt by
analyzing the sequences of 106 viruses isolated there from birds
and humans between 2006 and 2009: 85 sequences were obtained
from the National Center for Biotechnology Information (NCBI)
database, and 21 sequences were newly obtained in this study. At
the time of this investigation, these 106 sequences represented
40% of the complete and partial H5N1 virus sequences from
Egypt in public databases. HPAI H5N1 emerged in Egypt first in
poultry in 2006, swiftly spread to many species of birds in different
geographic regions [11,22], and was declared endemic in 2008
[11]. Human infections started shortly thereafter and reached 112
cases by October 2010 [1,10].
Phylogenetic analysis of the 106 H5N1 virus HA genes showed
that all of these HA genes clustered in clade 2.2.1, with some of
these viruses forming several new H5 sublineages (Figure 1). H5N1
isolates from 2006­2007 were interspersed throughout the
phylogenetic tree, indicating rapid spread of the ancestral HA
gene. In contrast, most human and avian isolates from 2008­2009
were clustered separately in distinct sublineages, denoted here as
sublineages A, B (I, II), C and D. These phylogenetic relationships
indicated that during 2007­2008 the genetic diversity of H5 HA in
Egypt increased dramatically and resulted in the establishment of
distinct human and avian sublineages. Conversely, phylogenetic
analysis of viral neuraminidase (NA) genes revealed that these
genes were less divergent (Figure S1), with branches and tree
topology different than the HA tree. The NA sequences formed a
single monophyletic cluster which included the virus with the
ancestral HA gene. These findings suggested that H5N1 viruses
circulating in Egypt have diversified without significant genetic
linkage, at least between the HA and NA genes.
SA binding specificity of H5N1 viruses isolated in Egypt
The phylogenetic distribution of human and avian isolates in
Egypt prompted us to investigate whether recent Egyptian isolates
had an altered receptor binding specificity. To determine the a2,3
SA- and a2,6 SA-binding affinity of these isolates, we performed
direct binding assays with SAa2,3Gal and SAa2,6Gal sialylglyco-
polymers [23,24]. Six H5N1 isolates from outbreaks in Egypt
during 2007­2009 were tested: A/duck/Egypt/D1Br12/2007
(EG/D1), A/chicken/Egypt/C1Tr13/2007 (EG/C1), A/chicken/
Egypt/RIMD11-1/2008 (EG/11), A/chicken/Egypt/RIMD12-3/
2008 (EG/12), A/chicken/Egypt/RIMD28-1/2009 (EG/28), and
A/chicken/Egypt/RIMD29-3/2008 (EG/29). EG/D1 and EG/
C1 were isolated from 2007 outbreaks, shared .99% homology
with H5N1 viruses isolated in 2006, and in our phylogenetic tree did
not form a sublineage or group with other H5N1 viruses isolated in
Egypt, implying that they emerged before the establishment of new
sublineages in Egypt and indicating that they were phylogentically
close to the original H5N1 genotype in Egypt. The other four
isolates belonged to the new H5 phylogenetic sublineages (Figure 1),
indicating that they emerged during more recent H5N1 outbreaks.
Preliminary experiments to determine optimal binding assay
conditions showed the importance of using appropriate virus
titers (i.e., hemagglutination titers), because high virus titers
produced exaggerated signals for the weakly binding glycopoly-
mer (a2,6 SA) and low titers only detected binding to the high-
affinity glycopolymer (a2,3 SA) (Figure S2). For example, EG/
Author Summary
Even though highly pathogenic avian H5N1 influenza
viruses lack an efficient mechanism for human-human
transmission, these viruses are endemic in birds in China,
Indonesia, Viet Nam and Egypt. Hotspots for bird-human
transmission are indicated in areas where human cases are
more than 80% of total H5N1 influenza cases. Circulation
among hosts may allow H5N1 virus to acquire amino acid
changes enabling efficient bird-human transmission and
eventually human-human transmission. The receptor
specificity of viral hemagglutinin (HA) is considered a
main factor affecting efficient transmissibility. Several
amino acid substitutions in H5 virus HAs that increase
their human-type receptor specificity have been described
in virus isolates from patients, but their prevalence has
been limited. In contrast, we show here that new H5
sublineages in Egypt have acquired a change in receptor
specificity during their diversification in birds. We found
that viruses in those sublineages exhibited increased
attachment and infectivity in the human lower respiratory
tract, but not in the larynx. Our findings may not allow a
conclusion on the high pandemic potential of H5N1 virus
in Egypt, but helps explain why Egypt has recently had the
highest number of human H5 cases worldwide.
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D1, which was expected to have a classical avian influenza virus
a2,3 SA specificity, showed strong binding to a2,3 SA as
expected, but also measurable binding to a2,6 SA when the virus
titer was increased to 512 HAU. Conversely, EG/12, which was
assumed to have increased a2,6 SA specificity because it
clustered with human sublineage A strains, showed a complete
loss of a2,6 SA binding with increasing dilution of the HA titer to
8 HAU. From these results, HA titers from 32 to 128 HAU
appeared to be optimal for comparison of receptor binding
specificity with our experimental conditions. Therefore, HA
titers of all virus samples were adjusted to an HA titer of 64
HAU, relative to a reference EG/D1 sample, and used for the
following binding assays.
EG/D1, EG/C1, EG/11, EG/28 and EG/29 viruses had
binding specificity for a2,3 SA (Figure 2C­2G). The association
constants are shown in Table S1. The binding patterns closely
resembled the strong a2,3 SA binding specificity observed with an
avian influenza H5N3 virus, A/Duck/Hong Kong/820/80
(Figure 2B). In contrast, EG/12 virus had appreciably increased
binding to a2,6 SA, with binding to both a2,3 SA and a2,6 SA
(Figure 2H). However, the EG/12 binding affinity for a2,6 SA was
less than that of the seasonal human influenza virus A/Japan/
434/2003 (Figure 2A). This was confirmed by direct binding
assays using recombinant viruses generated by reverse-genetics:
each recombinant virus contained one of the HA genes in a
background of all of the other EG/D1 virus genes (denoted here as
rEG/D1) (Figure 2I­2M). To investigate other sublineage A and B
viruses, we synthesized the HAs of three H5N1 viruses isolated in
Egypt: a bird isolate; A/goose/Egpt/0929-NLQP/2009 (EG/
0929); and two human isolates; A/Egypt/N04822/2009 (EG/
4822) and A/Egypt/N02039/2009 (EG/2039). The receptor
specificities of these viruses were determined and showed that
the H5 HAs of these recent isolates also had increased a2,6 SA
binding (Figure 2N­2P). These results indicated differences in HA
affinity to a2,6 SA among recent H5 isolates, together with an
affinity to a2,3 SA.
Identification of amino acid mutations in viral HAs
enabling a2,6 SA binding
To identify mutations enabling a2,6 SA binding, we focused on
viruses in sublineages A and B, to which most human isolates
belonged. Comparison of 6 HA sequences of sublineage A viruses
with 100 HA sequences of other H5 viruses isolated in Egypt
identified two amino acid changes in the sublineage A virus HAs
(Table 1): Q192H and S235P (H5 HA numbering). Introduction
of the Q192H mutation into EG/D1 HA (denoted rEG/D1Q192H)
markedly increased viral binding to a2,6 SA (Figure 3A).
However, introduction of the S235P mutation into EG/D1 HA
(denoted rEG/D1S235P) only slightly increased a2,6 SA binding.
There was no synergistic effect with both mutations: the double
Figure 1. Phylogenetic tree of HA genes of H5N1 viruses
isolated in Egypt. This tree includes published HA sequences of 85
H5N1 influenza A viruses isolated in Egypt, from the National Center for
Biotechnology Information database (minimum sequence length
1,644 nt), and 21 HA sequences determined in this study (sequence
length 1,707 nt). The newly analyzed sequences in this study are
marked with a black circle. The strains whose HA sequences were
determined in this study and were analyzed further for receptor binding
specificity are marked with a red circle. The strains whose HA sequences
were previously reported and were analyzed for receptor binding
specificity in this study are marked with a blue circle. Colors are used to
highlight virus strains with different hosts, isolation year and
sublineage.
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mutant had similar a2,6 SA binding to that of the single Q192H
mutant. In contrast, the H192Q mutation, but not the P235S
mutation, in HAs of EG/12 (denoted rEG/D1-EG/12 HAH192Q)
and EG/4822 decreased a2,6 SA binding (Figures 3B and S3A).
These findings suggested that the Q192H mutation in H5N1 avian
viruses increased the binding affinity of HA for the human
receptor.
The HA sequences of the 19 H5N1 viruses in sublineage BI
(denoted sublineage A1 in a previous report [25]) differed from the
87 HA sequences of other H5N1 viruses isolated in Egypt at three
Figure 2. Receptor-binding specificity of H5N1 viruses isolated in Egypt. Direct binding of viruses to sialylglycopolymers containing either
a2,3-linked (blue) or a2,6-linked (red) sialic acids was measured. (A) Seasonal human influenza H3N2 virus. (B) Avian influenza H5N3 virus. (C)­(H)
Isolates from 2007­2009 outbreaks in Egypt. (I)­(P) Recombinant EG/D1 viruses with different HAs as indicated. Each data point is the mean 6 SD of
triplicate experiments.
doi:10.1371/journal.ppat.1002068.g002
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HA amino acid residues: S120N, 129 deletion (D) and I151T
(Table 1). When introduced as a single mutation into EG/D1
HA, none of these amino acid changes increased binding
to a2,6 SA (Figure 4A). However, the 129D/I151T double
mutation increased a2,6 SA binding. In contrast, both the 129S
insertion and the T151I mutation in the HAs of EG/0929 and
EG/2039 decreased a2,6 SA binding (Figures 4B and S3B). These
results suggested that the 129D and I151T mutations acted
synergistically to enable a2,6 SA binding by sublineage BI viruses.
Sublineage BII viruses have four mutations: the three mutations
found in sublineage BI viruses plus an additional V210I mutation
(Table 1). When introduced as a single mutation in the HA of EG/
D1, V210I partially increased a2,6 SA binding, but there was not
an appreciable increase in binding in the V210I/129D/I151T
triple mutant (Figure 5). These results suggested that the V210I
mutation did not increase a2,6 SA binding in sublineage BII
viruses above that of the 129D/I151T double mutation.
Genetic properties of H5N1 viruses in sublineages A, BI
and BII
The phylogenetic trees of sublineages A, BI and BII suggested
human viruses in these sublineages emerged from avian viruses in
these sublineages or closely related avian viruses (Figure 1). The
amino acid changes in HA enabling a2,6 SA binding in sublineage A
(Q192H) and sublineage BI viruses (129D/I151T) were not found in
human H5N1 influenza viruses phylogenetically unrelated to
sublineage A and B strains (data not shown), indicating that these
mutations were associated with the phylogeny of avian H5N1
sublineage A and B viruses in Egypt. A database search of virus gene
sequences posted since 2006 also revealed that the prevalence of
these amino acid changes increased in human influenza virus HAs in
Egypt concurrently with an increase in avian influenza viruses in
Egypt (Table 2), although most of the recent avian influenza virus
isolates were in sublineages C and D (Figure 1). In contrast, an
increased prevalence has not been detected in either birds or humans
in Asia. These findings suggested that H5N1 avian viruses in Egypt
acquired binding affinity for a2,6 SA during viral diversification in
local bird populations, which may have contributed to subsequent
virus transmission to humans with higher efficiency.
Attachment of rEG/D1 viruses in the human respiratory
tract
To investigate whether mutations in avian virus HAs enabling
a2,6 SA binding function with similar specificity in the human
respiratory tract, the attachment pattern of selected viruses to fixed
tissues of the human upper and lower respiratory tract (i.e., larynx,
trachea and alveoli) was determined by histochemistry. Histo-
chemical analysis can provide clinically relevant data on virus
attachment in human airway epithelia [26,27] and on the glycan
topologies that influenza viruses target for cell-specific infections in
airway epithelia [28,29]. Human H3N2 virus, which was used as a
control, attached extensively to ciliated epithelial cells in the larynx
and trachea and, to lesser degree, to alveolar cells (type I
pneumocytes; Figure 6). In contrast, rEG/D1, rEG/D1-EG/11
HA and rEG/D1-EG/29 HA attached predominantly to alveolar
cells (type II pneumocytes), with little attachment in larynx and
trachea, as found for avian H5N3 virus. The attachment pattern of
rEG/D1-EG/12 HA was different from the classical avian pattern
found for H5N3: little attachment to larynx, moderate attachment
to trachea, and significant attachment to alveoli (both type I and II
pneumocytes). The attachment patterns of the rEG/D1Q192H,
rEG/D1129D,I151T and rEGD1129D,I151T,V210I mutants were sim-
ilar to that of rEG/D1-EG/12 HA. However, all three mutant
viruses attached less abundantly to trachea than the H3N2 virus.
Also, rEG/D1-EG/12 HAH192Q showed an attachment pattern
similar to that of rEG/D1, with rare attachment to trachea. We
also performed virus histochemistry on sialidase-treated sections,
which abrogated all staining confirming that the viruses in this
study did not bind to non-sialic acid residues (Figure S4). Although
not quantitative, these results indicated that mutations enabling
a2,6 SA binding are clinically significant in affecting the affinity of
HA for receptors in the human respiratory tract.
Replication of rEG/D1 viruses in a human airway
epithelial culture
To examine whether the HA mutations enabling a2,6 SA
binding also affected virus replication in human airway cells, we
studied virus growth in primary human small airway epithelial
cells (SAEC) by infecting these cells with selected recombinant
viruses and human H3N2 virus, which was used as a control, at a
multiplicity of infection (MOI) of 1 or 0.1 and monitoring viral
growth kinetics and cytopathicity for 72 h post-infection. For
comparison, we studied viral growth kinetics in chicken embryo
fibroblast (CEF) cells infected at an MOI of 0.1 or 0.01. All viruses
replicated well in CEF cells and produced .107
focus-forming
units (FFU)/ml at 24 and 48 h post-infection. The difference in
titers of these viruses was ,1 log FFU/ml at each time point,
indicating that all of the viruses replicated equally well in avian-
Table 1. Mutations in HA genes in H5 viruses in sublineages A, BI and BII.
Sublineage (no. of strains
in sublineage) Isolation year Mutation in HAa
% of strains with mutation (no. of strains with
mutation/total no. of strains)
A (6) 2008­2009 Q192H 100 (6/6)
S235P 100 (6/6)
BI (19) 2007­2009 S120N 94 (18/19)
129D 100 (19/19)
I151T 100 (19/19)
BII (5) 2009 S120N 100 (5/5)
129D 100 (5/5)
I151T 100 (5/5)
V210I 100 (5/5)
a
H5 numbering, D denotes deletion.
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derived cells (Figure 7A). These results confirmed that there was
no incompatibility between EG/12 HA and EG/D1 NA or
between mutated EG/D1 HA and EG/D1 NA in the recombi-
nant viruses generated for this study (compare the kinetics of
parental EG/D1 and rEG/D1 viruses in Figure 7A). In contrast,
in SAEC cells (Figure 7B), rEG/D1Q192H, rEGD1129D,I151T and
rEG/D1-EG/12 HA replicated more efficiently than rEG/D1
and rEG/D1-EG/12 HAH192Q, with slight differences in their
growth, and a final virus titer of rEG/D1Q192H . rEG/
D1129D,I151T . rEG/D1-EG/12 HA. These viruses replicated in
SAEC cells and reached titers more similar to those of human
H3N2 virus than of parental EG/D1, especially at a higher
inoculum. The difference in virus growth kinetics correlated with
cytopathicity in SAEC cells: rEG/D1Q192H, rEG/D1129D,I151T
and rEG/D1-EG/12 HA produced more severe cytopathic effects
and resulted in more detachment of infected cells at 24, 48 and
Figure 3. Effect of HA mutations in sublineage A viruses on receptor specificity of EG/D1 virus HA. (A) The two mutations found in the
HAs of sublineage A viruses were introduced into the HA of EG/D1 virus as single and double mutations. (B) The reverse mutations were introduced
into the HA of EG/12 virus. Direct binding to sialylglycopolymers containing either a2,3-linked (blue) or a2,6-linked (red) sialic acid was assayed.
Mutations are indicated by subscripts. Each data point is the mean 6 SD of triplicate experiments.
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72 h post-infection than rEG/D1 and rEG/D1-EG/12 HAH192Q
(Figure 7C). These results indicated that the Q192H mutant and
the 129D/I151T double mutant produced a substantial viral
growth advantage in human airway epithelial cells.
Effect of HA mutations on virulence of rEG/D1 viruses in
mice in vivo
To assess the effect of enhanced a2,6 SA binding on
pathogenicity of H5N1 isolates from Egypt, BALB/c mice were
inoculated intranasally with different dilutions of selected recom-
binant viruses. Mice inoculated with 36104
FFU rEG/D1Q192H,
rEG/D1129D,I151T or rEG/D1-EG/12 HA showed considerable
weight loss (Figure 8A). In contrast, mice inoculated with 36104
FFU rEG/D1 or rEG/D1-EG/12 HAH192Q showed no clinical
effects during the 14 d observation period, and most mice infected
with 36105
FFU of these viruses survived. The lethality of rEG/
D1Q192H, rEG/D1129D,I151T and rEG/D1-EG/12 HA was
substantially higher: the MLD50 was 8.86102
FFU for rEG/
D1Q192H, 1.56103
FFU for rEG/D1129D,I151T and 1.36104
FFU
for rEG/D1-EG/12 HA (Figure 8B), .50 times less than the
MLD50 of 5.96105
FFU for both rEG/D1 and rEG/D1-EG/12
HAH192Q. Consistent with this result, the virus yield in lungs of
mice infected with 36104
FFU of the three viruses was .10-fold
higher 4 d post-infection and .110-fold higher 7 d post-infection,
and at a dose of 36105
FFU was .70-fold higher 4 d post-
infection than with parental rEG/D1 virus (Figure 8C).
Lungs of mice infected with 36104
FFU viruses were examined
by histopathology at 7 d post-infection. Mice infected with rEG/
D1Q192H, rEG/D1129D,I151T or rEG/D1-EG/12 HA had much
more dramatic pathological changes in their pulmonary airways
and parenchymal tissues. The lungs had moderate to severe
bronchiolar necrosis and alveolitis with associated hyperplasia,
pulmonary edema and inflammatory cell infiltrates (Figure 9C­
9E). In contrast, lung pathology of rEG/D1 and rEG/D1-EG/12
HAH192Q infected mice showed signs of limited lymphohistiocytic
cell extravasations (Figure 9B and 9F). Mock-infected mice did not
have lesions in their lungs (Figure 9A). H5 antigen was more
extensively detected by immunohistochemistry in the alveolar area
of lungs infected with rEG/D1Q192H, rEG/D1129D,I151T or rEG/
D1-EG/12 HA than in lungs infected with rEG/D1 and rEG/D1-
EG/12 HAH192Q (Figure 9G­9L). Weak antigen staining was only
rarely detected in the bronchiolar area in lungs of mice infected
with rEG/D1 and rEG/D1-EG/12 HAH192Q (see insert in
Figure 9H and 9L). Therefore, the difference in lethality in mice
Figure 4. Effect of HA mutations in sublineage BI viruses on receptor specificity of EG/D1 HA. (A) The mutations found in sublineage BI
viral HAs were introduced as single and multiple mutations into the HA of EG/D1 virus. (B) The reverse mutations were introduced into the HA of EG/
0929 virus. Direct binding to sialylglycopolymers containing either a2,3-linked (blue) or a2,6-linked (red) sialic acid was measured. Mutations are
indicated by subscripts. Each data point is the mean 6 SD of triplicate experiments.
doi:10.1371/journal.ppat.1002068.g004
Figure 5. Effect of HA mutations in sublineage BII viruses on receptor specificity of EG/D1 HA. The mutations found in sublineage BII
viral HAs were introduced as single and multiple mutations into the HA of EG/D1 virus. Direct binding to sialylglycopolymers containing either a2,3-
linked (blue) or a2,6-linked (red) sialic acid was measured. Mutations are indicated by subscripts. Each data point is the mean 6 SD of triplicate
experiments.
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infected with these viruses was grossly correlated with the growth
kinetics and cytopathicity of the viruses in human airway epithelial
cells. Collectively, these results indicated that enhanced receptor
specificity in vivo enabled rEG/D1Q192H, rEG/D1129D,I151T and
rEG/D1-EG/12 HA to infect mice at lower titers than rEG/D1
and rEG/D1-EG/12 HAH192Q.
Effect of mutations on structural changes in EG/D1 HA
To investigate the structural basis for the changes in human
receptor-binding specificity in viruses in the new sublineages, we
generated models of the HA structures of EG/D1, EG/D1Q192H
and EG/D1129D,I151T from the crystal structure of the HA of A/
Vietnam/1194/04 (H5N1) (Protein Data Bank ID (PDBID) code
2IBX) [24], and performed a docking study with these models and
two types of ligands, SAa2,3Gal (PDIBID code 1MQM) and
SAa2,6Gal (PDIBID code 1MQN). In our modeling, HA residues
120, 210 and 235 were distant from the receptor binding sites in
the EG/D1 HA structure, whereas residues 129, 151 and 192
were located around them (Figure 10A and 10B). A Gln192 to
histidine mutation (and a Gln192 to arginine mutation) generated
a positively-charged side chain in the HA carbon backbone at this
position, which has been reported [24] to stabilize contact of
SAa2,6 Gal-terminated polysaccharides with H5 HA by forming a
hydrogen bond with human receptor moieties (also see Discussion
below). In addition, deletion of Ser129 led to a hydrogen bond
between side chains of the HA carbon backbone at Glu127 and
Thr151, affecting orientation of the 130-Loop (Figure 10C).
Therefore, the double 129D/I151T mutation might affect the
contact angle between human-type receptor ligands and viral HA.
In our simulation, the Udock scores of the complexes between the
SAa2,6 human-type receptor ligand and EG/D1, EG/D1Q192H
and EG/D1129D,I151T HA were 213.51, 218.05 and 216.48 kcal/
mol, respectively (Figure 10C). Therefore, the Udock scores of the
complexes bound to EG/D1Q192H and EG/D1129D,I151T HA were
more negative than with parental EG/D1 HA, indicating more
energetically stable interactions of the mutant HAs with the human
receptor analog. In contrast, the Udock scores of the complexes
between the SAa2,3 avian-type receptor ligand and EG/D1, EG/
D1Q192H and EG/D1129D,I151T HA were relatively similar (214.19,
215.27 and 212.16 kcal/mol, respectively), with the Udock scores
of the complexes bound to EG/D1Q192H and EG/D1129D,I151T HA
not appreciably more negative than with EG/D1 HA. These results
indicated that HAs of the viruses in the new sublineages have
structurally and energetically more stable conformations for binding
human receptors.
Discussion
In this study of H5N1 avian and human influenza viruses
isolated in Egypt, we found that these viruses clustered in several
new H5 sublineages, with a higher than expected binding affinity
for a2,6 SA, and identified the amino acid mutations responsible
for this expanded receptor specificity. Our phylogenetic analyses
also indicated that these viruses emerged during 2007­2008
outbreaks in Egypt. This time overlaps with or slightly precedes an
increase in the number of human cases of H5N1 virus infection in
Egypt [1,10].
HA plays an important role in the attachment of influenza
viruses to host cells and, therefore, influences viral host range and
pathogenicity [12,30­32]. In this study of H5N1 virus (clade
2.2.1), we found that an HA Q192H single mutation or a 129D/
I151T double mutation increased viral binding to a2,6 SA and
increased infection in human airway epithelia. Previous assays [24]
of A/Vietnam/3028II/04 virus (clade 1) and A/chicken/Indone-
sia/N1/05 (clade 2.1) binding to sialylglycopolymers found that an
HA Q192R mutation enhanced binding to a2,6 SA. The Q192H
mutation identified in this study was at the same residue as the
Q192R mutation in the A/Vietnam/3028II/04 and A/chicken/
Indonesia/N1/05 viruses, suggesting that these two mutations
produced a similar conformational change in HA. These structural
changes agreed fairly well with simulation data that the mutation
at this position in an H5 HA model electrostatically enhanced HA
binding affinity to human-like glycan [33]. The Q192H mutation
was not present in H5 HAs in 375 avian influenza viruses and 120
human influenza viruses isolated in Asia, including clade 1, 2.1,
2.2 and 2.3 viruses (Table 2). We examined codon usage in HA of
495 H5 isolates from Asia and 254 isolates from Egypt with Q at
residue 192 and found that all of these viruses encode 192Q using
codon CAA. This result indicates that an amino acid change from
Table 2. Prevalence of HA mutations characteristic of H5 sublineages A, BI and BII viruses in virus isolates from Egypt and Asia.
% of strains showing mutation characteristic of sublineage isolated in (no. of virus strains analyzed)a,b
Asia (495) Egypt (260)
Birds (375) Humans (120) Birds (189) Humans (71)
Isolation year (no. strains) Isolation year (no. strains) Isolation year (no. strains) Isolation year (no. strains)
Sub-
lineage
Characteristic
mutation
in HA
2006
(181)
2007
(134)
2008
(47)
2009
(13)
2006
(95)
2007
(20)
2008
(5)
2009
(0)
2006
(37)
2007
(70)
2008
(73)
2009
(9)
2006
(16)
2007
(23)
2008
(7)
2009
(25)
A Q192H 0 0 0 0 0 0 0 - 0 0 1.3 11.1 0 0 0 16.0
S235P 93.9 98.5 97.8 100 100 100 100 - 0 1.4 5.4 11.1 0 4.3 42.8 24.0
BI S120N 0.5 3.7 0 0 0 0 16.6 - 0 11.4 5.4 11.1 0 21.7 28.5 84.0
129 D 0 0 0 0 0 0 0 - 0 8.5 5.4 22.2 0 21.7 28.5 84.0
I151T 0 0.7 10.6 0 2.1 0 0 - 0 8.5 5.4 22.2 0 21.7 28.5 84.0
BII V210I 1.6 2.2 21.2 0 0 0 0 - 0 0 0 0 0 0 0 20.0
a
Percent of H5N1 viruses that have mutation characteristic of sublineages A, BI and BII for each geographic region, host and year.
b
Sequence information from the National Center for Biotechnology Information database in addition to sequences analyzed for this study.
- denotes no sequence information available.
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Figure 6. Attachment of rEG/D1 viruses to tissues of the human respiratory tract. The attachment patterns of A/Japan/434/2003 (H3N2), A/
Duck/Hong Kong/820/80 (H5N3), and eight rEG/D1 viruses (rEG/D1, rEG/D1-EG/11 HA, rEG/D1-EG/29 HA, rEG/D1-EG/12 HA, rEG/D1Q192H, rEG/
D1129D,I151T, rEG/D1129D,I151T,V210I and rEG/D1-EG/12 HAH192Q) to fixed human larynx, trachea and alveoli tissue sections were examined by
histochemistry. Attached viruses were stained red. Arrows and arrow-heads indicate type I and type II pneumocytes, respectively. The panels were
chosen to reflect the attachment pattern in each tissue section as much as possible.
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Q to H or R at residue 192 required a one nucleotide change
(CAA to CAT/CAC (H) or CGA(R)). The higher frequency of a
transversion to encode H may have enabled such a mutation to
occur more frequently in HAs of H5 viruses in Egypt.
Since the HA Q192R substitution might be selected during viral
growth in a human patient and enhance a2,6 SA binding in the
human respiratory tract [24], we constructed rEG/D1 with this
HA substitution and found its a2,6 SA binding affinity similar to or
slightly greater than rEG/D1Q192H and rEG/D1129D,I151T (data
not shown). In addition, deletion of HA residue 129 was not found
in any of the H5 HAs of the 495 Asian isolates examined, and the
I151T substitution was only detected in H5 HAs isolated from 6
birds and 2 human patients in Asia (1.6% and 1.7% prevalence,
respectively). H5 HA residues 129 and 151 make atomic contact
with sialoglycosides [34]. We showed here that the 129D mutation
generates a new hydrogen bond between Glu127 and Thr151,
resulting in conformational changes around the binding pocket
(Figure 10C). This effect around the glycosidic bond in H5 HAs
seems to be unique to the viruses isolated in Egypt. We also
searched for similar mutations (Q192H and the double 129D/
I151T mutation) in 4507 avian influenza viruses with HAs H1­13
and H16 and found that only 3 bird isolates had these mutations
(Table S2): Quail/Nanchang/12-340/2000 (H1N1), Turkey/
Minnesota/40550/1987 (H5N2), and Ruddy turnstone/Dela-
ware/2762/1987 (H11N2). Such mutations were not present in
any of the H1, H2 or H3 HAs of the human isolates in the early
years of the Spanish flu (1918), Asian flu (1957), Hong Kong flu
(1968) and Russian flu (1977) pandemics, in which these avian
subtypes crossed the species barrier to humans. However, it is
noteworthy that most of the viruses in this study that clustered in
sublineage B were reported to have evolved towards an H1N1-like
receptor usage, to efficiently replicate in the upper respiratory
Figure 7. Growth kinetics of rEG/D1 viruses in avian cells and human cells. (A) CEF cells were infected in triplicate with parental EG/D1 and
five rEG/D1 viruses (rEG/D1, rEG/D1Q192H, rEG/D1129D,I151T, rEG/D1-EG/12 HA and rEG/D1-EG/12 HAH192Q) at an MOI of 0.1 or 0.01. (B) Human SAEC
cells were infected in triplicate with the viruses at an MOI of 1 or 0.1. The culture supernatants were harvested at the indicated times and assayed for
focus-forming units on CEF cells to determine the progeny virus titer (log10 FFU/ml). Each data point in (A) and (B) is the mean 6 SD of triplicate
experiments. (C) Phase contrast microscopy of morphological changes in SAEC cells infected by the indicated viruses at an MOI of 0.1 and examined
at the indicated times post-infection.
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tract, and that structural properties of the receptor binding sites of
Spanish flu viruses and sublineage B viruses are much closer to
each other than to other H1N1 and H5N1 viruses [35]. In
contrast, a conformational change in HA due to S235P and
S120N mutations was not observed in our structural model: these
were also shown not to increase HA affinity for a2,6 SA by direct
binding assays (data not shown).
Our data suggested that H5N1 viruses from Egypt had acquired
amino acid mutations enabling a2,6 SA binding during their
transmission among birds, not during viral growth in human
patients. First, avian isolates were at the base and within branches
of the phylogenetic tree of new sublineages A and B, and clustered
closely with human isolates (Figure 1). Moreover, all of the avian
isolates already had identical mutations that contributed to
Figure 8. Mortality and weight loss of mice infected with rEG/D1 viruses. Six-week-old BALB/c mice (7­8 mice per group) were inoculated
intranasally with the indicated doses of rEG/D1, rEG/D1Q192H, rEG/D1129D,I151T, rEG/D1-EG/12 HA and rEG/D1-EG/12 HAH192Q. (A) Body weight of
infected mice was monitored up to 14 d post-infection. Mean percent body weight change (6SD) for each group of mice is shown. (B) Survival of
mice inoculated with rEG/D1 viruses. Mortality was calculated including mice that were sacrificed because they had lost more than 30% of their body
weight. (C) Virus titers in lungs of mice infected with 36104
or 36105
FFU rEG/D1 at the indicated times post-infection. Circles and diamonds indicate
values in individual mice.
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binding affinity for the human-type receptor (Tables S3, S4, S5).
Second, critical amino acid mutations involved in a2,6 SA
recognition (Q192H and the double 129D/I151T mutation) were
not found in any of the H5 HAs from human isolates
phylogenetically unrelated to sublineages A and B. Therefore, it
is unlikely that viruses with these mutations were newly selected
during viral growth in humans. Third, all viruses examined here
exhibited a classical avian a2,3 SA binding affinity and replicated
efficiently in CEF cells, suggesting that these viruses had retained
HAs for efficient transmission among birds (Figure 7A).
Several amino acid mutations that increase a2,6 SA binding
affinity of H5 virus HAs have recently been described in human
isolates [23,24,36]. It is possible that such mutations were selected
in humans and played an important role in viral recognition of
human-type receptors. However, there have been only limited
reports of those mutations in H5 HAs in infections in human
patients [23,24,36]. Considering that the mutations in H5 HAs in
birds identified here have been found in some population of birds
in the vicinity of humans, such viral mutations emerging in birds
may be as important risk factors for human H5N1 infections as
those mutations emerging in viruses infecting humans. Thus far,
there have been few reports of HPAIV in bird populations with
increased affinity for a2,6 SA [37].
At present, the determinants of efficient human-human
transmission by avian influenza viruses are not completely
understood [21,38]. It is generally thought that both a change in
receptor specificity from a2,3 SA to a2,6 SA and the resultant shift
in infection to the upper respiratory tract are essential [39].
However, most amino acid mutations in H5 HAs that have been
reported to increase a2,6 SA binding have not conferred a
complete change in receptor specificity in the original virus genetic
background [23,24,40,41]. But, Chutinimitkul et al. have recently
reported that some mutations can cause a complete change in the
A/Indonesia/5/05 background [42]. Increased a2,6 SA binding
affinity and reduced a2,3 SA binding affinity was also observed
among North American lineage H7 viruses isolated in 2002­2004
[37]. In contrast, we found that all of the H5N1 viruses in this
study retained the classical avian a2,3 SA binding affinity
(Figures 2­5). Histochemistry using human tissues also found that
viruses in this study with avian H5 HA mutations had little
attachment to the larynx, but moderate attachment to trachea and
abundant attachment in alveoli (Figure 6), whereas human H3N2
virus extensively bound to both the larynx and trachea. These
results suggested that H3 and H5 viruses recognized more
complex glycan topologies, which have not yet been fully
elucidated in human airway epithelia [28,29]. This conclusion is
in agreement with the suggestion that H5N1 viruses attach to
receptors in the human upper respiratory tract that are not
detected by lectin histochemistry and with data that H5N1 viruses
can productively replicate in ex vivo cultures of human
nasopharyngeal tissues [43].
These findings suggest that currently circulating H5N1 viruses
in Egypt lack gene products for efficient human-human
transmission, even though they have caused a relatively large
number of human cases in Egypt. Indeed, most human infections
resulted from direct exposure to H5N1 virus-infected poultry or
poultry products and no sustained human-human transmission
has been documented to date in Egypt [1,44]. It should be noted
that our findings do not allow determination of the potential for
an H5N1-derived pandemic virus in Egypt. However, the
emergence of sublineage A and B H5N1 viruses is a possible
contributing factor to Egypt recently having the highest number
of human H5N1 influenza virus cases in the world, with repeated
avian infections increasing the probability of avian-human
transmission. To our knowledge, this is the first report identifying
amino acid changes in H5 HA responsible for an increase in
human H5N1 infections in an endemic area.
Mice have been an animal model for studying influenza
[45­47]. In this study, we found that the HA mutations enabling
a2,6 SA binding enhanced viral virulence in BALB/c mice
(Figure 8). These results are consistent with a previous report on
different influenza viruses and different HA amino acid residues
Figure 9. Histopathology and immunohistochemistry in lung
tissues of mice infected with rEG/D1 viruses. Photomicrographs
of hematoxylin-and-eosin (H&E) stained and immunohistochemically
(IHC) stained lung sections from mice infected with 36104
FFU rEG/D1
viruses 7 d post-infection are shown as follows. (A) and (G) mock-
infected. (B) and (H) rEG/D1-infected. (C) and (I) rEG/D1Q192H-infected.
(D) and (J) rEG/D1129D,I151T-infected. (E) and (K) rEG/D1-EG/12 HA-
infected. (F) and (L) rEG/D1-EG/12 HAH192Q-infected. In the IHC-stained
tissues, viral antigen is stained deep brown on a hematoxylin-stained
background (arrows). In mice infected with rEG/D1 and rEG/D1-EG/12
HAH192Q, positive staining was detected sporadically in the bronchiolar
epithelium (insert).
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in a ferret model [48]. Reports on lectin histochemistry showed
that BALB/c mice express both a2,3 and a2,6 SA in airway
epithelia, with a2,3 SA specifically expressed in the upper
respiratory tract and a2,6 SA expressed in pulmonary paren-
chyma [49]. Previous histochemistry report on seasonal human
influenza viruses H3N2 and H1N1 showed rare attachment to
mouse type I pneumocytes, indicating the presence of glycan
topologies in alveoli to which influenza viruses with a2,6 SA
binding affinity attach [27]. In this study, recombinant avian H5
viruses with single and double point mutations that should affect
receptor binding were found to have acquired a2,6 SA binding
affinity, and the resultant expansion of receptor specificity in vivo
contributed to enhanced virulence in mice. Indeed, virus titers in
the lungs of mice infected with the mutant viruses were more
than one log higher than in mice infected with the parental virus
(Figure 8C), corresponding to severe histopathological changes
(Figure 9). These results were consistent with histochemistry
showing that the mutants acquired enhanced attachment affinity
to human type I pneumocytes (Figure 6). Type I pneumocytes
comprise 96% of the alveolar surface area, which is extremely
thin, thereby minimizing the diffusion distance between the
alveolar air space and pulmonary capillary blood [50].
Therefore, viral binding specificity for this cell type has
implications for the development of pneumonia. However, other
factors also need to be considered, such as the low similarity of
the SA expression pattern in mice relative to that in humans
[26,27]. Thus, it would be of interest to determine the effect of
the substitutions in HA described here on virus virulence in the
ferret model, which is a more suitable animal model for human
H5N1 viral pneumonia [26,27]. Our studies also found that EG/
D1, an ancestral strain of currently circulating H5N1 viruses in
Egypt, was not highly pathogenic in mice, as indicated by an
MLD50 .105
FFU (Figure 8B). Avian and human H5N1 viruses
in Egypt, including EG/D1, encode PB2-627Lys, which
reportedly enhances the host range and virulence of influenza
viruses [30,47,51]. The results of this study indicate that this
Figure 10. Analysis of receptor docking modes of EG/D1 HA and HA mutants. Structural models of H5 HA. (A) Ribbon model of EG/D1 HA.
The trimeric globular-head region is shown. Key residues in our analysis are shown in a colored space-filling model. Receptor binding domains are
colored blue (130 loop), green (190 helix) and purple (220 loop). (B) Molecular surface of EG/D1 HA. The red circle indicates the receptor binding
pocket. (C) Docking models for EG/D1, EG/D1Q192H and EG/D1129D,I151T HA with a human-type receptor analog (PDBID code 1MQN). Residues 127E,
128A, 130S and 131G are colored green, as is 129S, and the other residues and domains are displayed in the same colors as above. An additional
hydrogen bond between E127 and T151 is indicated in the red circle. The Udock scores of the corresponding complexes are shown at the bottom.
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amino acid residue alone does not provide sufficient replicative
advantage in mammals for the influenza viruses (clade 2.2.1) in
Egypt, although it may be a prerequisite for H5N1 virus
virulence in mammalian hosts.
The mechanism underlying the emergence of H5N1 viruses in
Egypt with both a2,3 SA and a2,6 SA binding affinities is
unclear. Some H7 viruses isolated in North America from 2002­
2004 showed a marked decrease in a2,3 SA binding together with
increased binding to glycans with a2,6 SA [37], and several
H5N1 field isolates (clade 2.3.4) in the Lao People's Democratic
Republic in 2007­2008 had reduced binding to a2,3 SA
receptors [52]. In contrast, H5 viruses isolated in Egypt have
retained the classical avian a2,3 SA binding affinity (Figures 2­5).
Previous studies have shown passage of H5N1 viruses through
land-based poultry as a possible mechanism for emergence of
dual receptor specificity [17,53]. However, most bird isolates in
Egypt, found to be clustered in sublineages A and B in this study,
were recently reported to be derived from domestic waterfowl,
not from land-based poultry [54]. In addition, these H5N1
viruses showed an appreciably different attachment pattern in the
human respiratory tract than that of typical avian viruses
(Figure 6). Therefore, the binding properties of H5 viruses in
Egypt may be the result of geographic and cultural factors that
have yet to be identified.
Egypt has a relatively large number of human cases of H5N1
virus infection, and the highest number of cases worldwide since
2009 [1,10]. The influenza virus phylogenetic tree suggests that
sublineages A and B, the focus of this study, emerged during virus
diversification in birds. At present, viruses grouped in sublineages
C and D are widely disseminated across Egypt. Therefore, it
remains possible that repeated circulation in birds would allow
sublineage C and D viruses to acquire amino acid change(s) other
than those identified here that could enable increased a2,6 SA
binding affinity, although the amino acid mutations identified here
may be useful markers in assessing H5N1 field isolates for their
potential to infect humans. Since clade 2.2 appeared in Egypt in
2006, Egypt has had a single known introduction of a clade 2.2.1
H5N1 virus. Neither introduction of other phylogenetically
distinct sublineages of clade 2.2 (I, II and III) nor reassortment
events between the sublineages, as detected in neighboring Nigeria
[55­57], have been documented in Egypt [7,58]. Such events also
were not observed in our phylogenetic analyses of HA and NA
genes (Figures 1 and S1). However, introduction of these
sublineages into Egypt could accelerate the evolutionary dynamics
of H5N1 virus. Moreover, all Egyptian viruses (clade 2.2.1), which
emerged during the 2005 Qinghai Lake outbreak in China [3,4],
have mammalian-type PB2-627Lys [30,47,51], implying the
potential for evolution to a pandemic virus. Therefore, there is a
critical need for continued surveillance of birds to monitor
receptor specificities of H5N1 field isolates in Egypt as well as
the pandemic potential of these strains.
Materials and Methods
Ethics statement
All animal studies were conducted under the applicable laws
and guidelines for the care and use of laboratory animals in the
Research Institute for Microbial Diseases, Osaka University,
approved by the Animal Experiment Committee of the Research
Institute for Microbial Disease, Osaka University, as specified in
the Fundamental Guidelines for Proper Conduct of Animal
Experiment and Related Activities in Academic Research
Institutions under the jurisdiction of the Ministry of Education,
Culture, Sports, Science and Technology, Japan, 2006.
Virus isolation and preparation
During outbreaks of highly pathogenic avian influenza in Egypt
from January 2007 to February 2009, 27 nasopharyngeal swab
and tissue samples (lung and trachea) were collected from sick or
dead chickens and ducks from commercial farms and backyard
farms. Of these samples, 21 were identified as H5-positive by
reverse transcription-polymerase chain reaction (RT-PCR) and
selected for virus isolation. Twenty viruses were eventually isolated
by single passage in the allantoic cavity of 11-day-old embryonated
chicken eggs. The allantoic fluids were then harvested and stored
as seed viruses at 280uC. Laboratory strains A/Duck/Hong
Kong/820/80 (H5N3) and human influenza A virus A/Japan/
434/2003 (H3N2) were kindly provided by Yoshinobu Okuno,
Kanonji Institute, The Research Foundation for Microbial
Diseases of Osaka University, Kagawa, Japan. For subsequent
studies, allantoic fluids were pre-cleared by centrifugation at
3,000 rpm for 20 min and filtration through 0.45 mm filters, and
viruses were then purified by centrifugation at 25,000 rpm for 2 h
through 20% and 60% sucrose. After collection of the virus-
containing fractions, viruses were suspended in PBS and pelleted
by centrifugation at 25,000 rpm for 2 h. Virus pellets were
resuspended in PBS and aliquots were stored as working stocks at
280uC. Virus titers were assayed as FFU by focus-forming assays
[59] on CEF cells for avian influenza viruses and on MDCK cells
for human H3N2 virus. All experiments with live H5N1 viruses
were performed in Biosafety Level 3+ (BSL 3+) conditions at
Osaka University, as approved for work with these viruses by the
Ministry of Agriculture, Forestry and Fisheries, Japan.
Cells
CEF cells were prepared from 11-day-old embryonated eggs.
MDCK cells were purchased from the Riken BioResource Center
Cell Bank (http://www.brc.riken.jp/lab/cell/english/). These cell
lines were maintained in Dulbecco's Modified Eagle's Medium
supplemented with 10% heat-inactivated fetal calf serum at 37uC
in a humidified atmosphere of 95% air and 5% CO2 as described
previously [60]. Human primary SAEC cells were purchased from
the Lonza Corporation (http://www.lonza.com/) and maintained
according to the manufacturer's recommendations.
Sequence analysis
Viral RNA was extracted from viruses using Trizol Reagent
(Invitrogen, http://www.invitrogen.com/) according to the man-
ufacturer's protocol. RT-PCR was done using an oligonucleotide
(Uni12) complementary to the conserved 39 end of viral RNA
[61]. Gene cloning and sequencing were done on at least 3
independent clones per segment as described previously [62].
The nucleotide sequence data analyzed for viruses in this study
are available in the DDBJ/EMBL/GenBank databases under the
accession numbers AB601121 to AB601156.
Generation of viruses by reverse genetics
Recombinant viruses were generated with a plasmid-based
reverse genetics system [63]. The viral complementary DNAs were
cloned into pUC18-based plasmids, between the human RNA
polymerase I promoter and the hepatitis delta virus ribozyme
(pPOLI). All viruses generated by reverse genetics carried the HA
gene of one of the viruses being studied, with the other genes
coming from EG/D1. The HA genes of EG/0929, EG/4822 and
EG/2039 were synthesized using the sequences registered in the
NCBI database Influenza Virus Resource (IVR, http://www.ncbi.
nlm.nih.gov/genomes/FLU/FLU.html) and site-directed muta-
gensis PCR (GeneTailor Site-Directed Mutagenesis System;
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PLoS Pathogens | www.plospathogens.org 15 May 2011 | Volume 7 | Issue 5 | e1002068
Invitrogen). Mutant HA genes were generated by PCR-based site-
directed mutagenesis in the EG/D1, EG/12, EG/0929, EG/4822
or Eg/2039 HA background. All constructs were sequenced
completely to ensure the absence of unwanted mutations.
Recombinant viruses were generated by plasmid transfection of
co-cultured 293T and CEF cells, and were propagated in eggs.
The HA genes of the virus stocks were sequenced to detect the
possible emergence of revertants during amplification.
Genetic analysis
For phylogenetic analysis of HA genes, published HA sequences
of 85 representative H5N1 influenza A viruses isolated in Egypt
from 2006 to 2009 were obtained from the NCBI database
(http://www.ncbi.nlm.nih.gov/nucleotide). Phylogenetic analysis
was performed on those 85 HA sequences and on the HA
sequences of the 21 viruses isolated in this study using MEGA4
software [64] for the neighbor-joining method, with the nucleotide
sequences covering most of HA gene. Estimates of the phylogenies
were calculated by performing 1,000 bootstrap replicates. For
phylogenetic analysis of NA genes, published NA sequences from
the NCBI database of 65 representative H5N1 viruses isolated in
Egypt from 2006 to 2009 together with the NA sequences of 19
viruses isolated in this study were analyzed. For a database search,
published sequences of 260 HA genes from influenza A viruses
isolated in Egypt from 2006 to 2009 from NCBI IVR were
analyzed. For comparison, published HA sequences of 495 H5N1
influenza A viruses recently identified in Asia were also obtained
from NCBI IVR. These sequences were aligned by the MAFFT
program [65] and the HA1 regions were compared with the
sequences of the viruses isolated in this study.
Hemagglutination titration
Stocks of avian and human influenza viruses were serially
diluted with PBS and mixed with 0.5% chicken red blood cells
and 0.75% guinea pig red blood cells, respectively. Hemagglu-
tination by avian and human influenza viruses was observed
after incubation at room temperature for 30 min or 1 h,
respectively to determine their HAU. To correct for differences
in HAU values due to different blood lots, a reference virus
sample was used and HAU values of all virus samples were
adjusted relative to the reference HAU titer of EG/D1, which
was used in the optimization analysis of the following receptor
specificity assay.
Receptor specificity assay
Receptor binding specificity was analyzed by a solid-phase
direct binding assay as described previously [23,24,52], with a
sialylglycopolymer containing N-acetylneuraminic acid linked to
galactose through either an a2,3 or a2,6 bond (Neu5Aca2,3-
LacNAcb-pAP, and Neu5Aca2,6LacNAcb-pAP). Serial dilutions
of each sialylglycopolymer were prepared in PBS, and 100 ml was
added to each well of 96-well microtiter plates (Polystyrene
Universal-Bind Microplates, Corning, http://www.corning.com/).
The plates were then irradiated with 254 nm ultraviolet light for
10 min and each well was washed three times with 250 ml PBS.
Each well was blocked with 100 ml PBS containing 0.1% Tween 20
(PBST) and 2% bovine serum albumin at room temperature for 1 h.
After washing with ice-cold PBST, a solution containing influenza
viruses (64 HAU in PBST) was added to each well and the plates
were incubated at 4uC for 12 h. After washing five times with ice-
cold PBST, mouse anti-NP antibody (against influenza virus NP
protein) was added to each well and the plates were incubated at
4uC for 2 h. The wells were then washed five times with ice-cold
PBST and incubated with peroxidase-conjugated goat anti-
immunoglobulin (Histofine Simple Stain MAX-PO, Nichirei,
http://www.nichirei.co.jp/bio/english/) at 4uC for 2 h. After
washing five times with ice-cold PBST, 100 ml premixed tetra-
metylbenzidine-H2O2 substrate was added to each well. After
incubation at room temperature for 10 min, the reactions were
stopped with 50 ml 1 M H2SO4, and absorbance at 450/630 nm
was measured.
Binding data were plotted against the concentration of sialic
acid residues in the reaction solution and were analyzed using
GraphPad Prism version 5.0 (GraphPad Software, http://www.
graphpad.com/). To determine the apparent association con-
stant (Ka) values, nonlinear regression was used to fit the data
based on the one-site model. Each data point is the mean 6 SD
of three to six experiments, which were each performed in
triplicate.
Viral growth kinetics in SAEC and CEF cells
SAEC cells were infected in triplicate with the indicated viruses
at an MOI of 1 or 0.1. The virus inoculum was removed after 1 h
and the cells were washed and airway epithelial growth medium
(SAGM; Lonza) containing bovine pituitary extract (BPE; 30 mg/
ml), hydrocortisone (0.5 mg/ml), human epidermal growth factor
(hEGF; 0.5 ng/ml), epinephrine (0.5 mg/ml), transferrin (10 mg/
ml), insulin (5 mg/ml), triiodothyronine (6.5 ng/ml), bovine
serum albumin-fatty-acid free (BSA-FAF; 50 mg/ml), retinoic
acid (RA; 0.1 ng/ml), gentamycin (30 mg/ml) and amphotericin
B (15 ng/ml) was added. Acetylated trypsin (2 mg/ml, Sigma-
Aldrich, http://www.sigmaaldrich.com/) was also added to
SAEC cultures for propagation of human H3N2 virus. At the
indicated times post-infection, virus titers in the cell culture
supernatants were determined in triplicate by FFU assays in CEF.
For determination of viral growth in CEF cells, the cells were
infected in triplicate at an MOI of 0.1 or 0.01. At the indicated
times post-infection, virus titers were determined in triplicate by
FFU assays. Preliminary lectin-based flow cytochemistry studies
indicated a difference in SA expression on the surface of SAEC
and CEF cells without growth under air-liquid interface
conditions, with predominant expression of a2,6 SA in SAEC
cells and of a2,3 SA in CEF cells. Therefore, all cell cultures in
this study were established without air-liquid interface conditions
as described previously [47,66].
Virus histochemistry in tissue sections
To produce fluorescein isothiocyanate (FITC)-labeled viruses
for histochemistry, influenza viruses, purified and concentrated as
described above, were inactivated with formalin in PBS (0.025%
final concentration) for 24 h at 37uC. The virus mixture was then
dialyzed against PBS for 18 h at 4uC and complete inactivation
was confirmed by assay on MDCK cells. A 1 ml sample of
inactivated virus was then mixed with 0.1 ml 1.1 M carbonate-
bicarbonate buffer (pH 9.5) containing 0.55 mg FITC isomer I
(Invitrogen)/ml for 1 h at room temperature with constant
stirring, followed by dialysis of the mixture against PBS for 42 h
at 4uC. To check for hemagglutination activity by the inactivated
virus, the viral hemagglutination titer was assayed after formalin
inactivation and FITC labeling.
Formalin-fixed paraffin-embedded human respiratory tract
tissue sections were obtained from US Biomax, Inc. (http://
www.biomax.us/). The paraffin-embedded tissues were deparaffi-
nized with xylene and hydrated using graded alcohols. After
blocking with Carbo-Free Blocking Solution (Vector Laboratories,
http://www.vectorlabs.com/), the tissues were then blocked with
Blocking Reagent (Perkin Elmer, http://www.perkinelmer.com/).
FITC-labeled influenza viruses were incubated with tissue sections
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at 4uC for 12 h at a titer of 128 HAU per section. The FITC
label was detected with peroxidase-conjugated rabbit anti-
FITC antibody (Dako, http://www.dako.com/). The signal was
amplified with a tyramide signal amplification system (Perkin
Elmer) according to the manufacturer's instructions. Peroxidase
was visualized with 3-amino-9-ethyl-carbozole (AEC+ Substrate
Chromogen, Dako), resulting in a bright red precipitate. Tissues
were counterstained with hematoxylin and embedded in Aquatex
(Merck Chemicals, http://www.merck-chemicals.com/). Omis-
sion of FITC-labeled virus was used as a negative control. The
specificity of the virus histochemistry was verified as follows. Tissue
sections, deparaffinized and hydrated as described above, were
treated with Arthrobacter ureafaciens sialidase (100 mU/ml, Nacalai
Tesque, http://www.nacalai.co.jp/) in sodium acetate buffer
(100 mM, pH 5.8) for 1 h at 37uC or mock-treated before
performing virus histochemistry. Micrographs were taken using a
Nikon Eclipse TE2000-U Inverted Microscope (Nikon, http://
www.nikon.com/).
Experimental infections in mice
To determine MLD50 values, groups of 6-week-old female
BALB/c mice (Japan SLC, Inc., http://www.jslc.co.jp/), under
isoflurane anesthesia, were inoculated intranasally with serial
10-fold dilutions of virus in 75 ml PBS, and MLD50 values were
calculated by the Reed-Muench method and expressed as FFU
required for 1 MLD50. Mice were observed daily for 14 d for
weight loss and mortality. Mice that lost .30% of their original
weight were euthanized. At 4 and 7 d after inoculation with
36104
FFU and at 4 d after inoculation with 36105
FFU
(because of mouse deaths before day 7 at this dose), virus titers
in the lungs were assayed as FFU in CEF cells. Virus titers in
lungs were expressed as log10 FFU. The lower limit of virus
detection was 2 log10 FFU/lung. For histopathology analysis,
mouse lungs collected at 7 d after inoculation with 36104
FFU
were fixed in 4% buffered paraformaldehyde, embedded in
paraffin, cut into 5 mm sections, stained with hematoxylin and
eosin, and examined by light microscopy. Immunohistochemical
staining for the H5 antigen was performed on deparaffinized
sections using a monoclonal antibody (C43) specific for the
nucleoprotein of influenza A virus by a two-step peroxidase
method (Hisfine Mouse Stain Kit, Nichirei) with diaminobenzi-
dine as the chromogen and hematoxylin as the counterstain. For
controls, unrelated antibodies were used in place of the primary
antibody.
Homology modeling and docking
The crystal structure of the HA of influenza virus A/Vietnam/
1194/04 (H5N1) (Protein Data Bank ID code 2IBX) [24] was used
as a template for homology modeling of EG/D1, EG/D1Q192H,
and EG/D129D,I151T by the Molecular Operating Environment
(MOE, http://www.chemcom.com). SA a2,3- and SA a2,6-linked
analogs (PDBID code 1MQM and 1MQN) were used as the input
for a docking study with the model HA structure using MOE
ASEDock [67]. The MMFF94x force field and the generalized Born
(GB) solvation model were used for the minimization step. The
complexes were evaluated by Udock scores which show the affinity
between ligand and receptor. Because SA a2,3- and SA a2,6-linked
analogs are a disaccharide and a trisaccharide respectively, the
absolute value of their Udock scores cannot be compared between
the complex bound to the a2,3-linked analog and that bound to the
a2,6-linked analog. However, Udock scores enable the binding mode
of the same analog to different HAs to be compared.
Supporting Information
Figure S1 Phylogenetic tree of NA genes of H5N1 viruses isolated
in Egypt. This tree includes published NA sequences of 63 H5N1
influenza A viruses isolated in Egypt, from the National Center for
Biotechnology Information database (minimum sequence length
1,150 nt), and 19 NA sequences determined in this study (sequence
length 1,350 nt). The sequences analyzed in this study are marked
with a black circle. Colors are used to highlight virus strains with
different hosts, isolation year and sublineage.
(TIF)
Figure S2 Optimization of viral HA titers for direct binding
assays. These assays were done using 4-fold dilutions of EG/D1
and EG/12 viruses (measured as HAU), with titers ranging from
512 to 8 HAU. Direct binding of viruses to sialylglycopolymers
containing either a2,3-linked (blue) or a2,6-linked (red) SA was
measured. Each data point is the mean 6 SD of triplicate
experiments.
(TIF)
Figure S3 Effect of reverse mutations in sublineage A and BI
virus HAs on receptor specificity. The reverse mutations to those
in Figures 3 and 4 were introduced into the HAs of sublineage A
virus EG/4822 (A) and sublineage BI virus EG/2039 (B). Direct
binding to sialylglycopolymers containing either a2,3-linked (blue)
or a2,6-linked (red) sialic acid was measured. Mutations are
indicated by subscripts. Each data point is the mean 6 SD of
triplicate experiments.
(TIF)
Figure S4 Specificity of virus histochemistry. Attachment of A/
Japan/434/2003 (H3N2), upper two panels, and EG/D1 virus,
lower two panels, to human respiratory tract tissues. Tissue
sections were treated or mock-treated with Arthrobacter ureafaciens
sialidase before performing virus histochemistry. The panels were
chosen to reflect the attachment pattern in each tissue section as
much as possible.
(TIF)
Table S1 Virus binding affinity to sialylglycopolymers.
(PPT)
Table S2 Virus strains encoding HA 129D/I151T and Q192H
mutations in avian influenza virus A virus subtypes.
(PPT)
Table S3 Properties of H5N1 influenza viruses in sublineage A.
(PPT)
Table S4 Properties of H5N1 influenza viruses in sublineage BI.
(PPT)
Table S5 Properties of H5N1 influenza viruses in sublineage
BII.
(PPT)
Acknowledgments
We thank K. Murata for the sequencing analyses; T. Nakaya, R. Kubota-
Koketsu, T. Daidoji, M Yasugi, Y. Inoue for valuable advice and
discussions; and A. Yamashita and T. Yasunaga for computational
assistance and resources.
Author Contributions
Conceived and designed the experiments: YW KI. Performed the
experiments: YW MSI NK RM. Analyzed the data: YW MSI NK RM
HH NS. Contributed reagents/materials/analysis tools: YW MSI HFE
NK HH NS TT YS. Wrote the paper: YW YS KI.
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PLoS Pathogens | www.plospathogens.org 17 May 2011 | Volume 7 | Issue 5 | e1002068
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Emergence of New H5N1 Influenza Virus Sublineages
PLoS Pathogens | www.plospathogens.org 19 May 2011 | Volume 7 | Issue 5 | e1002068
