Download Alterations in receptor-binding properties of swine influenza viruses

Journal of General Virology (2010), 91, 938–948
DOI 10.1099/vir.0.016691-0
Alterations in receptor-binding properties of swine
influenza viruses of the H1 subtype after isolation in
embryonated chicken eggs
Nobuhiro Takemae,1,2 Ruttapong Ruttanapumma,3 Sujira Parchariyanon,3
Shuji Yoneyama,4 Tsuyoshi Hayashi,1,2 Hiroaki Hiramatsu,5
Nongluk Sriwilaijaroen,5,6 Yuko Uchida,1,2 Sachiko Kondo,7,8
Hirokazu Yagi,8 Koichi Kato,7,8,9,10 Yasuo Suzuki5,11 and Takehiko Saito1,2
Correspondence
Takehiko Saito
[email protected]
1
Thailand–Japan Zoonotic Diseases Collaboration Center, Kasetklang, Chatuchak, Bangkok
10900, Thailand
2
Research Team for Zoonotic Diseases, National Institute of Animal Health (NIAH), National
Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki 305-0856, Japan
3
NIAH, Kasetklang, Chatuchak, Bangkok 10900, Thailand
4
Tochigi Central Livestock Hygiene Service Center, Utsunomiya, Tochigi 321-0905, Japan
5
Health Science Hills, College of Life and Health Sciences, Chubu University, Kasugai, Aichi
487-8501, Japan
6
Faculty of Medicine, Thammasat University (Rangsit Campus), Pathumthani 12120, Thailand
7
GLYENCE Co. Ltd, Aichi 464-0858, Japan
8
Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi 467-8603, Japan
9
Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes
of Natural Sciences, Aichi 444-8787, Japan
10
The Glycoscience Institute, Ochanomizu University, Tokyo 112-8610, Japan
11
Global COE Program for Innovation in Human Health Sciences, Shizuoka 422-8526, Japan
Received 13 September 2009
Accepted 9 December 2009
938
Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their
isolation in embryonated chicken eggs have not been well studied. In this study, the receptorbinding properties of classical H1 SIVs isolated solely in eggs or Madin–Darby canine kidney
(MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G
in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes
identical to those of the original viruses present in the clinical samples. Egg isolates with
substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and
sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with
D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay
using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac)
a2,6galactose (Gal) or a Neu5Aca2,3Gal linkage revealed that the egg isolates used in this study
showed higher binding activity to the Neu5Aca2,3Gal receptor than MDCK isolates. Increased
binding activity of the egg isolates to the Neu5Aca2,3Gal receptor was also confirmed by
haemagglutination assay with resialylated chicken erythrocytes by Galb1,3/4GlcNAca2,3sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of
the erythrocytes. The a2,3-linked sialic acids were expressed predominantly on the surface of
mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Aca2,3Gal more
abundantly than Neu5Aca2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic
acid (Neu5Gc) a2,6Gal and Neu5Aca2,6Gal. Our results demonstrate clearly that classical H1
SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in
the HA protein during isolation and propagation in embryonated chicken eggs.
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Receptor-binding properties of swine influenza viruses
INTRODUCTION
Swine influenza viruses (SIVs) have been recognized as an
important pathogen in swine production and a publichealth concern worldwide (Olsen et al., 2006a). Three
subtypes, H1N1, H1N2 and H3N2, have been predominantly circulating in pig populations over several decades
(Van Reeth, 2007). However, these subtypes have genetically distinctive characters in different geographical locations, and complicated genetic diversity has thus been
demonstrated even among the same subtypes (Brown et al.,
1998; Campitelli et al., 1997; Guan et al., 1996; Karasin
et al., 2006; Olsen et al., 2006b; Saito et al., 2008; Takemae
et al., 2008; Vincent et al., 2008; Webby et al., 2000;
Yu et al., 2009). Because of the segmented nature of the
genome, co-infection with more than two influenza viruses
of different origins in a single host allows viruses to
exchange their genes, resulting in this genetic diversity
(Olsen et al., 2006a).
Pigs are known to be susceptible to both human and avian
influenza viruses (Kida et al., 1994). Influenza viruses bind
to the sialic acid (SA) linked to galactose (Gal) on host cells
through the receptor-binding site of the haemagglutinin
(HA) protein (Lamb & Krug, 2001). Human viruses bind
preferentially to SA linked to Gal by an a2,6 linkage
(SAa2,6Gal), whereas avian viruses bind preferentially to
SAa2,3Gal (Rogers & D’Souza, 1989; Rogers & Paulson,
1983). It has been demonstrated that both the SAa2,3Gal
and SAa2,6Gal receptors are distributed on the pig tracheal
epithelium (Ito et al., 1998). This observation substantiates
the hypothesis that pigs can be a mixing vessel of human
and avian influenza viruses. In addition, SIVs with the
classical swine H1 gene have been reported to bind to
both 5-N-acetylneuraminic acid (Neu5Ac) a2,3Gal and
Neu5Aca2,6Gal (Matrosovich et al., 2000).
Two substrates, embryonated chicken eggs and Madin–
Darby canine kidney (MDCK) cells, have generally been
used for the isolation and propagation of influenza viruses
over the years (Lamb & Krug, 2001). Human viruses
isolated and grown in eggs, however, change their receptorbinding properties, which is known as egg adaptation (Katz
et al., 1987). Numerous studies have been carried out to
elucidate the mechanism of egg adaptation in human
H1N1 and H3N2 influenza viruses (Katz et al., 1987;
Robertson et al., 1987). Several passages of a human
influenza virus in the allantoic cavity of eggs induce amino
acid substitutions within the receptor-binding site on the
HA1 molecule, resulting in acquisition of specificity to
SAa2,3Gal receptors (Gambaryan et al., 1999; Ito et al.,
1997b). Such alteration of binding properties was suggested
to arise under selective pressure by the receptors on the
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are AB514929–AB514943.
A supplementary figure showing HPLC profiles of pyridylamino
derivatives of N-glycans derived from rabbit and chicken erythrocytes
is available with the online version of this paper.
http://vir.sgmjournals.org
cells in which the virus replicates; epithelial cells of the
allantoic cavity possess only receptors with the a2,3 linkage,
whereas MDCK cells possess receptors with both the a2,3
and a2,6 linkages (Ito et al., 1997b).
Unlike human influenza A viruses, alteration of the
receptor-binding specificities of SIVs after isolation and
passages in embryonated eggs has not been well documented. A recent study indicated that MDCK-grown SIVs
with classical swine H1 genes displayed affinity to sialyl
glycopolymers with a2,6 linkage, but not to those with a2,3
linkage, suggesting that egg-adapted SIVs altered their
receptor-binding properties (Gambaryan et al., 2005). The
present study was designed to clarify the effects of substrate
for the isolation of SIVs on their growth characteristics and
receptor-binding specificities. We investigated the amino
acid changes on the HA1 molecule associated with isolation
and passages in eggs by comparing putative amino acid
sequences obtained from clinical specimen-derived, egggrown and MDCK-grown SIVs of the H1N1 and H1N2
subtypes isolated in 2008 in Thailand and Japan,
respectively. The receptor-binding properties of egg-grown
and MDCK-grown viruses were compared by their
haemagglutinating activities with erythrocytes from different animal species. A receptor-binding assay using sialyl
glycopolymer and a haemagglutination assay using enzymically resialylated chicken erythrocytes were also performed. SA linkages on the erythrocyte surfaces were
examined by N-glycan and flow-cytometric analyses. The
results provide significant information on the receptorbinding properties of SIVs, information that substantiates
the egg adaptation of SIVs and enables us to suggest a
preferential substrate for isolating SIVs.
RESULTS AND DISCUSSION
Virus isolation and amino acid substitutions
observed in egg isolates
SIV of the H1N1 subtype, designated A/swine/Ratchaburi/
NIAH101942/2008 (Rat101942), was isolated from a nasal
swab collected from a clinically healthy, 12-week-old
fattening pig in both MDCK cells and the allantoic cavity
of embryonated eggs. After two passages in each substrate,
the supernatant of the infected cells showed HA activity at
a titre of 8, whereas infectious allantoic fluid showed HA
activity at a titre of 4, with 1 % guinea pig red blood cells
(RBCs). The MDCK isolate of A/swine/Tochigi/1/2008
(H1N2) (Tochigi1) was obtained in this study from the
lung homogenate of a 22-week-old pig in Japan that
showed coughing and a high body temperature (Yoneyama
et al., 2010). Harvested viruses from MDCK cells and from
the amniotic fluid of Tochigi1 after two passages in each
substrate showed HA titres of 4 and 16 with 1 % chicken
RBCs, respectively. However, Tochigi1 did not grow in
allantoic fluid in earlier passages (Yoneyama et al., 2010).
The passage histories of Rat101942 and Tochigi1 analysed
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939
N. Takemae and others
in this study appear in Table 1. Phylogenetic analysis of HA
gene sequences of both viruses obtained by direct
sequencing of the nasal swabs revealed that they belonged
to the classical swine H1 lineage (data not shown). N1 and
other internal genes of Rat101942 resided in the Eurasian
avian-like swine lineage (data not shown), as did previously
characterized Thai H1N1 isolates (Takemae et al., 2008).
Coding sequences of the MDCK isolates of Rat101942
(MD5) were identical in all eight gene segments to the
sequences obtained from PCR products amplified directly
from the specimen. This was also the case with the MDCK
isolate of Tochigi1 (MD2) in the HA, neuraminidase (NA)
and nucleoprotein (NP) genes. Sequence comparison
between MD5 and Al3 of Rat101942 revealed substitutions
in the deduced amino acid sequences at D190V (H3
numbering; Nobusawa et al., 1991) in the HA1 protein and
K99R in the NP protein (Table 1). Double peaks of A and
G at nt 665 in HA1, resulting in a mixed population (D
and G) at position 225 in the HA1 region, were seen in
Am2 of Tochigi1. Thus, MDCK isolates appear to represent
viruses originally existing in the specimens. The amino
acids at positions 190 and 225 in HA1 are known to be
located at the receptor-binding site and the left edge of the
receptor-binding pocket of the HA protein (Gamblin et al.,
2004; Nobusawa et al., 1991), respectively (Fig. 1).
It was interesting to note that, although valine predominated at position 190 in Al3 of Rat101942, a smaller peak of
A at nt 560 in HA1, resulting in a non-synonymous
substitution of D at position 190, was observed in the
chromatography of the sequence analysis. To examine the
extent of each variant of Rat101942 at the position, 50
clones encoding the HA1 regions from each sample (nasal
swab, MD5 and Al3) were analysed by using a TOPO TA
cloning kit for sequencing (Invitrogen) according to the
manufacturer’s instructions. Forty-seven of 50 clones
(94 %) had Val at position 190 in HA1 of Al3. No
variations were found in the clones derived from the nasal
swab and MD5 (Table 1).
Comparison of growth ability and receptorbinding properties between MDCK isolates and
egg isolates
The observation that egg isolates have an amino acid
substitution in the vicinity of the receptor-binding site in
the HA1 region prompted us to examine whether such
Table 1. Comparison of growth characteristics and nucleotide and amino acid sequences
NA,
Not applicable.
Virus/passage
history*
HA titreD
log10(EID50
ml”1)D
log10(TCID50
ml”1)D
log10(EID50)”
log10(TCID50)d
HA
NP
Nucleotide Amino acid§ Nucleotide Amino acid
A/Sw/Ratchaburi/NIAH101942/2008
NA
NA
Nasal swab
MDCK isolates
MD5
64
8.32
MD9
16
7.20
MD10
64
7.45±0.05
Egg isolates
Al3
256
8.02
Al8-1||
128
8.10±0.09
Al8-2||
256
8.43±0.38
A/Sw/Tochigi/1/2008
Lung
MDCK isolate
MD4
Egg isolate
Am2Al3
NA
NA
559
G
560
A
187(190)
D (100)
296
A
99
K
7.20
6.30
5.90±0.17
1.12
0.90
1.60a
G
G
G
A
A
A
D (100)
D
D
A
A
A
K
K
K
6.41
6.00±0.60
7.14±0.22
1.60
2.13b
1.28a
G
G
A
T/A
T
A
V(94)/D(6)
V
N
A/G
G
G
K/R
R
R
NA
NA
NA
665
A
222(225)
D
–
32
7.03±0.28
7.16±0.13
20.13
A
D
–
64
8.24±0.07
7.74±0.30
G
G
–
NA
0.50a
*MD, Al and Am represent MDCK cells, allantoic and amniotic cavities, the substrates in which virus was cultivated, respectively. Numbers after
each substrate indicate the number of passages in each substrate.
D1 % guinea pig RBCs were used. Mean±SD titres (n53) are shown for the viruses used in the receptor-binding assays.
dabStatistically significant differences between EID50 and TCID50, P,0.05 (aStudent’s t-test; bWelch’s t-test).
§H3 numbering according to Nobusawa et al. (1991). Values in parentheses after amino acid residues indicate percentages of clones with the
sequence shown.
||Different variants obtained during limiting dilutions.
No differences were found among the NP genes of A/Sw/Tochigi/1/2008.
940
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Journal of General Virology 91
Receptor-binding properties of swine influenza viruses
(a)
NP were observed in either MD9 passaged in eggs five
times (MD9Al5) or the egg isolates passaged five times on
MDCK cells (Al8-1MD5 and Al8-2MD5).
(b)
190
190
Tochigi1 MD2 was also propagated further twice in MDCK
cells (MD4), and Am2 in the allantoic cavity three times to
obtain Am2Al3 (Table 1). MD4 was confirmed to possess
HA, NA and NP genes identical to those seen in the lung
samples. Am2Al3 (D225G) showed a single peak of G at
nt 665 in HA1 in the chromatography of the sequence
analysis. Except for that residue, the identity of the HA, NA
and NP genes with Am2 was confirmed in Am2Al3.
225
225
Comparison of 50 % egg infective dose (EID50) ml21 and
50 % tissue culture infective dose (TCID50) ml21 values on
MDCK cells demonstrated that the Tochigi1 egg isolates
developed growth ability in the allantoic cavity of
embryonated eggs (Table 1). The virus titre in the eggs of
Tochigi1 Am2Al3 was significantly higher than the TCID50
ml21 value (Student’s t-test, P,0.05). The virus titre in the
eggs of Tochigi1 MD4 was comparable to that in the
MDCK cells. This suggests that the Tochigi1 egg isolates
were more adapted in their ability to replicate in the
allantoic cavity of embryonated eggs. Due to egg adaptation, there was increased growth ability of Tochigi1 Am2 in
embryonated eggs associated with D225G in the HA
molecule. In contrast, there was no change in the growth
abilities of Rat101942 in the allantoic cavity (Table 1). All
of the viruses obtained showed higher titres in the allantoic
cavity than in MDCK cells (Student’s t-test for MD10 and
Al8-2, P,0.05; Welch’s t-test for Al8-1, P,0.05). The
mechanism underlying the difference in growth ability in
embryonated eggs remains to be elucidated.
Fig. 1. Three-dimensional structure of the globular head of the HA
protein, based on A/swine/Iowa/30 (H1N1), from front (a) and
lateral (b) views. The locations of aa 190 and 225 are illustrated by
space-fill modelling. The receptor-binding site is shaded.
amino acid substitutions are associated with alteration(s)
in the receptor-binding properties and/or growth abilities
of the egg isolates.
Rat101942 strains were subjected to further plaque
purification in MDCK cells three times after MD5 and
limiting dilutions four times in eggs after Al3 to eliminate
the heterogeneity of the viruses before subjecting them to
analyses of their receptor-binding specificity. MD9 or
MD10, which were propagated on MDCK cells after plaque
purification, were confirmed to be identical in the HA gene
segment to the original virus. By the first limiting dilution,
two kinds of variant at the same position, D190N and
D190V, were obtained (Al4 of Rat101942). Clones possessing valine and asparagine at position 190 after final
limiting dilution followed by propagation were designated
Al8-1 (D190V) and Al8-2 (D190N), respectively (Table 1).
After establishing the clone, MD9 of Rat101942 was
passaged in the allantoic cavity of eggs, and the egg isolates
(Al8-1, Al8-2) were passaged in MDCK cells to examine
whether the substitutions at positions 190 in the HA1 and
99 in the NP were reversible in opposite environments. No
changes at position 190 in the HA1 or position 99 in the
Evidence that viruses derived from different substrates
differ in their receptor specificities was obtained by
haemagglutination tests with erythrocytes from different
animals (Table 2). MDCK and egg isolates agglutinated
rabbit, mouse and sheep erythrocytes differently. MDCK
isolates (MD10 of Rat101942 and MD4 of Tochigi1)
agglutinated rabbit erythrocytes with HA titres of 256 and
8, respectively. However, HA titres of the egg isolates
(Al8-1 and Al8-2 of Rat101942 and Am2Al3 of Tochigi1)
with rabbit erythrocytes were ,1. Although the MDCK
Table 2. Haemagglutinating activity with 1 % erythrocytes from different animals
HA titres of each virus were standardized to 64 by 1 % guinea pig RBCs. Amino acid substitutions in HA1 are indicated in parentheses. ,1 indicates
that no titres were observed with the undiluted viruses.
Virus/passage history
A/Sw/Ratchaburi/NIAH101942/2008
MD10 (190D)
Al8-1 (190DAV)
Al8-2 (190DAN)
A/Sw/Tochigi/1/2008
MD4 (225D)
Am2Al3 (225DAG)
http://vir.sgmjournals.org
Guinea pig
Chicken
Goose
Rabbit
Rat
Mouse
Sheep
64
64
64
32
32
32
32
32
32
256
,1
,1
16
64
32
,1
32
32
,1
16
16
64
64
4
32
8
8
8
,1
64
32
,1
2
,1
4
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941
N. Takemae and others
1.0
0.9 (a)
0.8
0.7
0.6
0.5
0.4
0.3
**
0.2
**
**
0.1 **
Rat101942 (MD10)
α 2,6
**
**
**
α 2,3
A450
10 20 30 40 50 60 70 80 90 100
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
(c)
Rat101942 (Al8-2)
α 2,6
*
*
*
*
α 2,3
10 20 30 40 50 60 70 80 90 100
1.0
(e)
Tochigi1 (Am2Al3)
0.9
0.8
α 2,6
**
**
0.7
**
0.6
**
α 2,3
0.5
0.4 * **
0.3 *
0.2
0.1 *
1.0
(b)
0.9
0.8
0.7
0.6
0.5
**
0.4
**
0.3 **
0.2
0.1
Rat101942 (Al8-1)
α 2,6
**
*
α 2,3
10 20 30 40 50 60 70 80 90 100
1.0
0.9 (d)
0.8
0.7
0.6
0.5
*
0.4
**
0.3
0.2 **
0.1
Tochigi1 (MD4)
**
*
α 2,6
**
α 2,3
10 20 30 40 50 60 70 80 90 100
Sialylglycopolymer (ng per well)
10 20 30 40 50 60 70 80 90 100
Sialylglycopolymer (ng per well)
isolates did not agglutinate mouse or sheep erythrocytes,
all egg isolates did (Table 2). Between Al8-1 and Al8-2
of Rat101942, no apparent differences were observed. In
addition, Am2Al3 of Tochigi1 had an 8-fold increase
in haemagglutination activity with chicken erythrocytes
compared with that of MD4 (Table 2). These results
revealed that the egg isolates had increased haemagglutinating activity with mouse and sheep erythrocytes
and reduced activity with rabbit erythrocytes after egg
adaptation.
A direct binding assay using sialyl glycopolymers demonstrated clearly that mutations at either position 190 or 225
in HA1 of egg isolates enhanced their Neu5Aca2,3Gal
specificity (Fig. 2). MD10 of Rat101942, possessing D at
position 190 in HA1, bound significantly to Neu5Aca2,6Gal,
but not to Neu5Aca2,3Gal (Student’s or Welch’s t-test
was applied according to preliminary tests of equality of
variances for concentrations from 1.6 to 100 ng per well,
P,0.01) (Fig. 2a). Increased binding to Neu5Aca2,3Gal
was demonstrated clearly with the egg-adapted isolates
of Rat101942, although they still bound preferentially to
Neu5Aca2,6Gal, as judged by the absorbance values from
the assay (Fig. 2b, c). The binding patterns obtained were
similar to that of the D225G mutation in HA1 of Tochigi1
(Fig. 2d, e). MD4 (225D) of Tochigi1 bound significantly to
Neu5Aca2,6Gal (Student’s or Welch’s t-test for concentrations except for 0 and 3.1 ng per well, P,0.05) (Fig. 2d),
942
**
Fig. 2. Direct binding assay using sialyl glycopolymers for Rat101942 and Tochigi1. The
receptor-binding activities of MD10 (a), Al8-1
(b) and Al8-2 (c) of Rat101942, and MD4 (d)
and Am2Al3 (e) of Tochigi1 are shown. h and
# represent mean±SD absorbances (n53)
of Neu5Aca2,3Gal and Neu5Aca2,6Gal, respectively. Asterisks indicate significant differences (*P,0.05; **P,0.01) between the
absorbances for each concentration of sialyl
glycopolymers by Student’s or Welch’s t-test.
whereas Am2Al3 (225G) clearly increased the binding
for Neu5Aca2,3Gal (Fig. 2e). These results showed that
Tochigi1 from a clinical sample could be grown successfully
in the amniotic cavity, but not in the allantoic cavity.
Amniotic membrane cells, which possess receptors with
both a2,3 and a2,6 linkages (Ito et al., 1997b), appeared to
allow Tochigi1 original virus with a2,6 specificity to replicate
in the amniotic cavity, whereas a minority of the Tochigi1
virus with the D225G substitution eventually grew to replace
the original population by utilizing a2,3 receptors. No
substitutions except for D225G were found in Tochigi1
Am2. Our previous study revealed that Japanese H1 SIVs
possessing 225D from clinical specimens did not grow well
in the allantoic cavity (Saito et al., 2008). A phenomenon
similar to that seen in the SIVs examined is well-known in
human influenza epidemic viruses. Human H3N2 viruses
passaged in the amniotic cavity several times showed a
substantial increase in a2,3 affinity, resulting from an amino
acid substitution (L226Q) in the receptor-binding site in the
HA1 molecule (Ito et al., 1997b). In addition, human H1
egg-adapted variants with either 190DAN or 225DAG/N
in the HA1 molecule always had increased affinity for
Neu5Aca2,3Gal-containing receptors (Gambaryan et al.,
1999).
The results described above provided us with another
perspective on residue 225 in the HA of classical H1 SIVs.
Most of the triple-reassortant H1 SIVs in North America,
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Journal of General Virology 91
Receptor-binding properties of swine influenza viruses
10
8
log2(HA titre)
in this study and some of the triple-reassortant H1 viruses
isolated from MDCK cells, such as A/swine/MN/22860-T/
2001 (H1N2), retained 225G (Choi et al., 2002). It is
therefore evident that not all D225G substitutions observed
in recent SIVs are due to egg adaptation.
Native CRBCs
2,3Gal R-CRBCs
2,6Gal R-CRBCs
6
4
2
MD10 Al8-1
Al8-2
(190D) (190V) (190N)
MD4 Am2Al3
(225D) (225G)
Rat101942
Tochigi1
Fig. 3. Haemagglutinating activity of Rat101942 and Tochigi1
with native CRBCs and R-CRBCs with Neu5Aca2,3Gal or
Neu5Aca2,6Gal.
as well as the H1N2 SIVs in Japan, have retained 225D
since 1999 and 2003 (Choi et al., 2002; Karasin et al., 2000;
Saito et al., 2008; Yassine et al., 2009), respectively, whereas
most of the classical SIVs circulating before the late 1990s
had 225G (Olsen et al., 2000; other data from GenBank). It
has been suspected that the classical swine H1 protein
retained 225G due to egg adaptation (Gambaryan et al.,
2005), coinciding with the fact that many laboratories used
embryonated eggs for SIV isolation before the 1990s (Kida
et al., 1994; Olsen et al., 2000). However, Rat101942 MD10
Chicken
Sheep
MDCK isolates (MD10 of Rat101942 and MD4 of
Tochigi1) appeared to possess an ability to bind to
Neu5Aca2,3Gal, because the binding of MDCK isolates to
Neu5Aca2,3Gal increased slightly at sialyl glycopolymer
concentrations of 12.5–100 ng per well when twice the
usual amount of MDCK isolates was used in the direct
binding assay (data not shown), suggesting that binding of
the MDCK isolates to Neu5Aca2,3Gal can be observed in
the assay when sufficient viruses against the receptors exist.
This was clearly supported by a haemagglutination assay
using resialylated chicken red blood cells (R-CRBCs)
(Fig. 3). MDCK isolates of both Rat101942 and Tochigi1
agglutinated the Neu5Aca2,3Gal R-CRBCs, although the
titres were lower than those for the native CRBCs and the
Neu5Aca2,6Gal R-CRBCs. All of the egg isolates were
shown to bind both receptors as well as those of native
CRBCs (Fig. 3), coinciding with the result in the direct
binding assay.
SA expression on RBCs
The relative amounts of SAa2,3Gal and SAa2,6Gal linkages
on the chicken, sheep, mouse and rabbit erythrocyte
surfaces, which showed different HA reactivity between
Mouse
Rabbit
Control
200
150
100
50
Counts
SA 2,3Gal
200
150
100
50
SA 2,6Gal
200
150
100
50
10
00 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
Fluorescence intensity
Fig. 4. Relative amounts of SAa2,3Gal and SAa2,6Gal on the surface of chicken, sheep, mouse and rabbit erythrocytes. Cell
numbers are plotted against log10(fluorescence intensity) of cells incubated without lectins (negative control) or with DIGlabelled lectins from Maackia amurensis (specific for SAa2,3Gal) or from Sambucus nigra (specific for SAa2,6Gal).
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N. Takemae and others
Table 3. Structures and relative quantities of the neutral, mono-, di- and trisialyl pyridylamino (PA) oligosaccharides derived from
rabbit and chicken erythrocytes
*Units of glucose (GU) were calculated from the elution times of the peaks obtained from the ODS column in Supplementary Fig. S1(b–e).
DRelative quantity (%) was calculated from the peak area in Supplementary Fig. S1(b–e) by comparison with total N-glycan content in rabbit and
chicken erythrocytes.
dStructures could not be identified by our analysis.
MDCK and egg isolates, reinforced the results described
above (Fig. 4). Chicken erythrocytes were labelled with
lectins binding to both SAa2,3Gal and SAa2,6Gal linkages,
as reported previously (Ito et al., 1997a). Sheep and mouse
erythrocytes were shown to contain the SAa2,3Gal linkage;
the presence of the SAa2,6Gal linkage was not demonstrated by this assay. In contrast, on rabbit erythrocytes, the
SAa2,6Gal linkage was shown to exist in a lower amount
and SAa2,3Gal linkage was not demonstrated.
N-glycan analysis characterized N-linked glycans expressed
on the surfaces of rabbit and chicken erythrocytes. Three
and four peaks were eluted by diethylaminoethyl columns
in rabbit and chicken erythrocytes, respectively [see
Supplementary Fig. S1(a), available in JGV Online]. Peaks
1, 2 and 3 observed in both erythrocyte types were
identified as neutral, monosialylated and disialylated Nglycans, respectively. Trisialylated glycan (peak 4) was only
observed in chicken erythrocytes. The molar ratios of each
944
N-glycan calculated from the peak areas were 83.5, 11.0
and 5.5 % in rabbit erythrocytes, respectively, and 28.9,
23.0, 26.5 and 21.6 % in chicken erythrocytes, respectively.
The N-glycan structure analysis of each peak obtained by
octadecyl silica (ODS) columns [Supplementary Fig. S1(b–
e)] identified 21 kinds of N-glycan, including 18 neutral
glycans, one monosialylated glycan and two disialylated glycans, on rabbit erythrocytes. Twenty-seven glycans, including ten neutral glycans, seven monosialylated
glycans, eight disialylated glycans and two trisialylated
glycans, were also identified on chicken erythrocytes. Each
structure identified is shown in Table 3. Both Neu5Ac and
5-N-glycolylneuraminic acid (Neu5Gc) were found on
rabbit erythrocytes, whilst, in contrast, only Neu5Ac
was found on chicken erythrocytes (Fig. 5; Table 3). The
most abundant SA species on rabbit erythrocytes was
Neu5Gca2,6Gal (8.2 %), which was about four times more
abundant than Neu5Aca2,6Gal (2.0 %; Fig. 5). On chicken erythrocytes, Neu5Aca2,3Gal (37.9 %) was expre-
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Journal of General Virology 91
3.18
Receptor-binding properties of swine influenza viruses
40
30
20
1.32
1.46
25
Rabbit
Chicken
0
5
0
10
Neu5Ac 2,3
Neu5Ac 2,6
Neu5Gc 2,3
Neu5Gc 2,6
Others
15
Neu5Ac 2,3 0.03
Neu5Ac 2,6 0.37
Neu5Gc 2,3 0
1.52
Neu5Gc 2,6
1.13
Others
Glycan content (%)
35
Fig. 5. Molar ratio of acidic N-glycans to total N-glycans on the
surface of rabbit and chicken erythrocytes. Each amount of acidic
N-glycans [pmol (mg dried erythrocytes)”1] is shown on the bars.
ssed more predominantly than Neu5Aca2,6Gal (17.4 %;
Fig. 5). The total amounts of N-glycans were 18.6 and
8.4 pmol (mg dried rabbit and chicken erythrocytes)21,
respectively.
The egg-adapted SIVs in this study did not agglutinate
rabbit erythrocytes (Table 2), which contain both Neu5Acand Neu5Gca2,6Gal molecules (Fig. 5), whereas the
MDCK isolates did. However, the egg-adapted isolates
agglutinated Neu5Aca2,6Gal R-CRBCs (Fig. 3). There
were four times more Neu5Gca2,6Gal molecules than
Neu5Aca2,6Gal molecules on rabbit erythrocytes (Fig. 5).
Neu5Gc molecules were shown to be expressed abundantly on pig tracheal epithelia (Suzuki et al., 1997).
Some of the avian-like H1 and human-like H3 swine
viruses, as well as the human H1 and H3 viruses, have
been suggested to recognize Neu5Gca2,6Gal molecules as
their receptors (Higa et al., 1985; Ito et al., 1997a). Those
observations suggest the potential of Neu5Gca2,6Gal
as a receptor for classical H1 SIVs. The mutation at
either residue 190 or 225 of HA1 during egg adaptation
might reduce the binding ability with Neu5Gca2,6Gal
receptors, so that the egg-adapted SIVs fail to agglutinate rabbit erythrocytes through Neu5Gca2,6Gal receptors. Further studies are needed to investigate the role
of Neu5Gca2,6Gal as a receptor molecule for classical
SIVs.
As has been pointed out by many researchers (Olsen et al.,
2006a), because pigs can act as a mixing vessel of influenza
viruses, it is crucial to monitor influenza viruses in these
animals to prevent emergence of a possible pandemic
virus (Webster et al., 1992). Cases of swine-to-human
transmission of influenza virus have been reported
http://vir.sgmjournals.org
sporadically worldwide many times in the past (Myers
et al., 2007). At least 11 sporadic human cases by triplereassortant swine H1N1(2) viruses were confirmed from
December 2005 to February 2009 in the USA (Shinde
et al., 2009). Pandemic (H1N1) 2009 viruses that possess
the HA, PB2, PB1, PA, NP and NS genes from triplereassortant H1 SIVs and the remaining two genes,
encoding the NA and matrix (M) proteins, from
Eurasian avian-like SIVs have been a threat to humans
since the infections of two patients were first identified in
April 2009 in the USA (CDC, 2009). Also, highly
pathogenic H5N1 avian influenza viruses still remain an
important public-health concern in the world (WHO,
2009). H5N1 viruses transmitted from poultry to pigs
have been isolated sporadically in China (Shi et al., 2008)
and Indonesia (Takano et al., 2009). It is crucial to
evaluate the receptor specificity of influenza virus isolated
from the pig population in order to assess transmission
ability among humans, predicting pandemic ability.
However, most of the swine H5N1 viruses in the abovementioned studies were isolated in embryonated eggs, and
thus could be subjected to egg adaptation by which their
ability to bind a2,6 linkages is altered. Even in a natural
setting, it has been demonstrated that the H1 avian virus
altered its receptor-binding property in pigs soon after
the epizootic of 1979 in the European swine population (Matrosovich et al., 2000). Choice of an adequate
substrate to isolate SIVs has therefore become increasingly
important for the detailed characterization of influenza
viruses isolated from pigs. Although a limited number of
strains were examined in the present study, the results
provide evidence that the MDCK cell is a more desirable
substrate than embryonated chicken egg for isolation of
influenza viruses from pigs.
METHODS
Sample collection and virus isolation. Forty nasal swabs were
collected from apparently healthy pigs, including 20 sows (1–2 years
old), ten 12-week-old fattening pigs and ten 9-week-old piglets, at a
farm in Ratchaburi province, Thailand, in January 2008. Each swab
was placed into a 15 ml tube containing 2 ml transport medium
[minimal essential medium (MEM; Invitrogen) including penicillin
(1000 units ml21), streptomycin (1000 mg ml21), fungizone
(25 mg ml21) and 0.5 % BSA, 0.01 M HEPES]. The swabs were
transported to NIAH, Thailand, on ice. Each swab was divided into
three portions. Two of the three portions were used for virus isolation
after filtering with a 0.45 mm pore size filter (Millipore): one was
inoculated into the allantoic cavities of 10-day-old embryonated
chicken eggs and incubated for 48 h at 37 uC, and the other was
inoculated onto a monolayer of MDCK cells for 45 min at 37 uC for
viral adsorption to the cells. After adsorption, MEM without fetal calf
serum containing 1 mg acetylated trypsin ml21 was added. The
inoculated cells were incubated for more than 48 h at 37 uC in 5 %
CO2 until the cytopathogenic effect appeared. Haemagglutinating
agents arising from the same swab in both an embryonated egg and
MDCK cells were identified as H1N1 by the PCR method using
specific primers designed in a previous study (Takemae et al., 2008).
They were designated A/swine/Ratchaburi/NIAH101942/2008
(H1N1) (Rat101942). The last portion was subjected to viral RNA
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N. Takemae and others
extraction by using an RNeasy mini kit (Qiagen), followed by RTPCR and direct sequencing of the PCR products. A lung specimen
from a pig, which yielded A/swine/Tochigi/1/2009 (H1N2) (Tochigi1)
(Yoneyama et al., 2010), was subjected to virus isolation in MDCK
cells at NIAH, Japan. After being ground in the transport medium
(10 %, w/v), the specimen was inoculated into MDCK cells as
described above. Tochigi1 isolated and passaged twice in the amniotic
cavity of embryonated chicken eggs at Tochigi Central Livestock
Hygiene Service Center was subjected to further analysis at NIAH,
Japan. Rat101942 and Tochigi1 were subsequently passaged in MDCK
cells or the allantoic cavity (Table 1).
Characterization of viruses and phylogenetic analysis. RT-PCR,
direct sequencing of the PCR products and phylogenetic analysis of
the viruses obtained in this study were carried out as described
previously (Takemae et al., 2008). The TCID50 ml21 values on MDCK
cells and the EID50 ml21 values at 37 uC of the viruses used were
determined.
Haemagglutination assay with erythrocytes of different animal
species. One per cent erythrocytes of guinea pig, chicken, goose,
rabbit, rat, mouse and sheep in PBS (pH 7.4) were used. When
comparing the haemagglutination activities of each virus with
different species, the quantities of each virus were standardized with
1 % guinea pig erythrocytes to give HA titres of 64. Viruses with HA
titres of ,64 were concentrated by using an Amicon Ultra centrifugal
filter with an MWCO 10 000 membrane (Millipore), then diluted
with PBS to obtain HA titres of 64 with 1 % guinea pig erythrocytes.
Titres were judged after 30 min incubation with chicken and goose
erythrocytes and 1 h incubation with the other mammalian
erythrocytes at room temperature (RT).
Direct binding assay. One hundred microlitres of serial dilutions of
sialyl glycopolymers containing Neu5Ac linked to Gal by either an
a2,3 or an a2,6 linkage in PBS were added to the wells of 96-flat-well
microplates (Costar 2504; Corning) to give a final concentration
range of 0–1000 ng ml21 (0–100 ng per well). The plates containing
sialyl glycopolymers were irradiated with UV light at 254 nm for
10 min and then washed three times with 300 ml PBS. The plates were
blocked with PBS Milk Blocking Solution (BioFX Laboratories) for
30 min at RT. After washing three times with PBS containing 0.1 %
Tween 20 (PBST), 50 ml virus-containing PBS with HA titres of 32
with 1 % guinea pig erythrocytes was applied to the wells and
incubated at 4 uC for 1 h. Virus-containing PBS was prepared to
reduce the materials binding non-specifically to sialyl glycopolymers
as follows: allantoic fluid or culture supernatant containing viruses
(MD10, Al8-1 and Al8-2 of Rat101942 and MD4 of Tochigi1) was
concentrated by using an Amicon Ultra centrifugal filter, then diluted
with PBS. Am2Al3 of Tochigi1 was partially purified by ultracentrifugation (28 000 r.p.m. for 2 h in an SW28 rotor) through 25 and
75 % sucrose, then diluted with PBS, because their non-specific
binding materials in allantoic fluid could not be removed by using the
Amicon Ultra centrifugal filter. After adsorption of the viruses to
sialyl glycopolymers, the plates were fixed with 100 ml 3.7 %
formaldehyde in each well for 30 min at RT. Allantoic fluid or
culture supernatant without viruses was used as a negative control
after filtration by an Amicon Ultra centrifugal filter. After five washes
with PBST, 50 ml anti-A/duck/Bavaria/2/1977 (H1N1) rabbit hyperimmune serum for Rat101942 strains and anti-A/swine/Iowa/15/1930
(H1N1) chicken hyperimmune serum for Tochigi1 at a dilution of
1 : 6400 with PBST containing 0.5 % BSA were added to respective
wells. These two different antibodies, prepared in NIAH, Japan, were
chosen by reactivity using the haemagglutination inhibition or ELISA
tests (data not shown). However, because of different affinities of the
antibodies to the viruses used, it was not possible to compare the
receptor-binding affinities of Rat101942 and Tochigi1. The plates
were incubated for 1 h at 37 uC. Next, after washing five times again
946
with PBST, 50 ml peroxidase-conjugated AffiniPure Goat Anti-Rabbit
IgG or peroxidase-conjugated AffiniPure Goat Anti-Chicken IgY
(IgG) (both from Jackson ImmunoResearch Laboratories) diluted at
1 : 64000 with PBST containing 0.5 % BSA was applied to the
respective wells. After incubation for 1 h at 37 uC, 100 ml tetramethylbenzidine substrate (BioFX Laboratories) was added to the
wells and the plates were incubated for 30 min at RT. Reactions were
stopped with 100 ml 450 nm Liquid Stop Solution (BioFX
Laboratories). Absorbances were measured at 450 nm.
Resialylation of CRBCs. CRBCs were modified enzymically to
contain exclusively either Neu5Aca2,3Gal or Neu5Aca2,6Gal. Briefly,
100 ml aliquots of 10 % CRBCs suspended with PBS(+) containing
0.9 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, were treated with 50 mU
sialidase from Vibrio cholerae (Roche), hydrolysing N- or O-acyl SAs
in a2,3, a2,6 and a2,8 bonds with glycoconjugates for 2 h at 37 uC.
Removal of SA from native CRBCs was confirmed by the absence of
haemagglutination activity with the SIVs used. Desialylated CRBCs
were washed twice with cold PBS, then divided into two aliquots and
resuspended in 50 ml PBS or PBS(+) containing 1 % BSA,
respectively. Cytidine 59-monophospho-N-acetylneuraminic acid
(1.5 mM; Sigma-Aldrich) with either 2.5 mU Galb1,4GlcNAca2,6sialyltransferase (Calbiochem) or 1 mU Galb1,3/4GlcNAca2,3-sialyltransferase (Calbiochem) was added into respective desialylated
CRBCs, then incubated at 37 uC for 4 h or overnight, respectively.
After three washes with cold PBS, the resialylated CRBCs were
suspended with 500 ml PBS at a final concentration of 1 % (v/v) and
then used for the haemagglutination assay. The HA titres were judged
after 1 h incubation at 4 uC.
Flow-cytometric analysis of SA linkages on erythrocytes.
Relative amounts of SAa2,3Gal and SAa2,6Gal on the surface of the
erythrocytes of chicken, sheep, mouse and rabbit were detected by
using a digoxigenin (DIG) glycan differentiation kit (Roche). Briefly,
approximately 56106 erythrocytes were washed twice with cold PBS
containing 10 mM glycine, followed by once with buffer 1 (50 mM
Tris/HCl, 0.15 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2,
pH 7.5). The washed erythrocytes were incubated with blocking
solution supplied with the DIG kit for more than 30 min at RT. DIGlabelled lectins, SNA (Sambucus nigra agglutinin) specific for
Neu5Ac/Neu5Gca2,6Gal residues or MAA (Maackia amurensis
agglutinin) specific for Neu5Ac/Neu5Gca2,3Gal residues, were
incubated at a final concentration of 1 mg ml21 with anti-DIG
antibodies conjugated to fluorescein (Roche) diluted 200-fold in
buffer 1 for 1 h at RT. Erythrocytes in blocking solution were
suspended in the lectin–antibody solution for 1 h at 4 uC. Control
experiments were performed without lectins. The fluorescence
intensity of 10 000 erythrocytes from each animal was analysed on a
flow cytometry EPICS XL system 2 (Beckman Coulter).
N-glycan
analysis
of
rabbit
and
chicken
erythrocytes.
Approximately 10 ml fresh blood obtained from rabbits and chickens
was haemolysed in 10 vols 0.2 % acetic acid, followed by three washes
with saline by centrifugation at 30 000 g for 15 min and then drying in
a vacuum overnight. Lipids of dried cells of rabbit (0.16 g) and chicken
(0.55 g) were extracted in a series of ethanol, chloroform/methanol and
acetone (Sriwilaijaroen et al., 2009). Subsequently, N-linked glycans of
each erythrocyte were analysed by multi-dimensional HPLC mapping
and matrix-assisted laser desorption–ionization time-of-flight mass
spectrometric (MALDI-TOF-MS) techniques as described previously
(Sriwilaijaroen et al., 2009; Takahashi & Kato, 2003).
ACKNOWLEDGEMENTS
This work was supported by the programme of the Founding
Research Center for Emerging and Reemerging Infectious Diseases
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Journal of General Virology 91
Receptor-binding properties of swine influenza viruses
commissioned by the Ministry of Education, Culture, Sports, Science
and Technology (MEXT) of Japan.
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Kawaoka, Y. & Webster, R. G. (1994). Potential for transmission of
avian influenza viruses to pigs. J Gen Virol 75, 2183–2188.
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