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Glycobiology Advance Access originally published online on March 27, 2007
Glycobiology 2007 17(7):713-724; doi:10.1093/glycob/cwm038
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza A viruses to human type receptors

Chao-Tan Guo3,4,5, Noriko Takahashi4,6,7, Hirokazu Yagi4,6, Koichi Kato4,6,7, Tadanobu Takahashi4,5, Shuang-Qin Yi8, Yong Chen3, Toshihiro Ito9, Koichi Otsuki10, Hiroshi Kida11, Yoshihiro Kawaoka4,12,13, Kazuya I-P Jwa Hidari4,5, Daisei Miyamoto4,5, Takashi Suzuki4,5 and Yasuo Suzuki1,2,4

2 Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, 11200 Matsumoto-cho, Kasugai-shi, Aichi 487-8501, Japan
3 Institute of Bioengineering, Zhejiang Academy of Medical Sciences, 182 Tianmushan Road, Hangzhou 310016, China
4 CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Japan
5 Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences, Suruga-ku, Shizuoka 422-8526, Japan
6 Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
7 GLYENCE Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464-0858, Japan
8 Department of Anatomy, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
9 Departments of Veterinary Public Health and
10 Veterinary Microbiology, Faculty of Agriculture, Tottori University, Tottori-shi, Tottori 680-8553, Japan
11 Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
12 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
13 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706

1 To whom correspondence should be addressed; Tel/Fax: +81 568 51 6391; e-mail: suzukiy{at}isc.chubu.ac.jp

Received on October 12, 2006; revised on March 13, 2007; accepted on March 22, 2007


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
The receptor specificity of influenza viruses is one factor that allows avian influenza viruses to cross the species barrier. The recent transmissions of avian H5N1 and H9N2 influenza viruses from chickens and/or quails to humans indicate that avian influenza viruses can directly infect humans without an intermediate host, such as pigs. In this study, we used two strains of influenza A virus (A/PR/8/34, which preferentially binds to an avian-type receptor, and A/Memphis/1/71, which preferentially binds to a human-type receptor) to probe the receptor specificities in host cells. Epithelial cells of both quail and chicken intestines (colons) could bind both avian- and human-type viruses. Infected cultured quail colon cells expressed viral protein and allowed replication of the virus strain A/PR/8/34 or A/Memphis/1/71. To understand the molecular basis of these phenomena, we further investigated the abundance of sialic acid (Sia) linked to galactose (Gal) by the {alpha}2-3 linkage (Sia{alpha}2-3Gal) and Sia{alpha}2-6Gal in host cells. In glycoprotein and glycolipid fractions from quail and chicken colon epithelial cells, there were some bound components of Sia–Gal linkage-specific lectins, Maackia amurensis agglutinin (specific for Sia{alpha}2-3 Gal) and Sambucus nigra agglutinin (specific for Sia{alpha}2-6Gal), indicating that both Sia{alpha}2–3Gal and Sia{alpha}2-6Gal exist in quail and chicken colon cells. Furthermore, we demonstrated by fluorescence high-performance liquid chromatography (HPLC) analysis that 5-N-acetylneuraminic acid was the main molecular species of Sia, and we demonstrated by multi-dimensional HPLC mapping and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis that bi-antennary complex-type glycans {alpha}2-6 sialylated at the terminal Gal residue(s) are major (more than 79%) sialyl N-glycans expressed by intestinal epithelial tissues in both the chicken and quail. Taken together, these results indicate that quails and chickens have molecular characterization as potential intermediate hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential.

Key words: influenza virus / receptor / sialic acid / chicken / quail


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Influenza viruses infect cells by binding of hemagglutinin (HA) to the sialyl sugar residue on the host cell surface (Suzuki 2005Go). Different sialic acid (Sia) species and linkages in various animals become a host barrier for influenza virus transmission among different hosts. Human epithelial cells in the respiratory tract have Sia{alpha}2-6galactose (Gal)-terminated glycoconjugates (human-type receptor) (Baum and Paulson 1990Go), while the intestines of ducks have Sia{alpha}2-3Gal-terminated glycoconjugates (avian-type receptor) (Ito et al. 1998Go; Suzuki 2005Go). Most isolated strains of influenza A viruses from humans preferentially recognize human-type receptors, whereas most avian strains preferentially recognize avian-type receptors. All known HA and neuraminidase (NA) subtypes of influenza A viruses were isolated from wild aquatic birds (Webster et al. 1992Go). Occasionally, avian influenza viruses are transmitted to other hosts such as pigs, horses, humans, and domestic poultry. The viruses implicated in the 1957 and 1968 pandemics were generated as the result of reassortment occurring between avian and human influenza viruses in pigs (Scholtissek et al. 1987Go; Schafer et al. 1993Go). In 1997, highly pathogenic H5N1 influenza viruses were transmitted directly from chickens to 18 humans, resulting in six deaths (Claas et al. 1998Go). Beginning in late 2003, outbreaks of highly pathogenic H5N1 viruses have been reported in Asian, European, and African countries (Enserink 2006Go; Webster et al. 2006Go), and transmission of the virus from birds to humans occurred in 12 countries, including Vietnam, China, Thailand, Indonesia, Cambodia, Turkey, Iraq, Egypt, Azerbaijan, Djibouti, Nigeria, and Lao People's Democratic Republic, and a total of 277 humans were infected by the virus, resulting in 167 deaths (World Health Organization, http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_03_01/en/index.html) up to March 1, 2007. In 1999, avian H9N2 influenza virus subtypes, which are widespread in poultry in Asia, were transmitted to two children (Peiris et al. 1999Go; Lin et al. 2000Go). These two virus isolates are similar to an H9N2 virus isolated from a quail in Hong Kong in late 1997. The recent transmission of avian H5N1, H7N7, and H9N2 influenza viruses from chickens and/or quails to humans indicate that avian viruses can directly infect humans without an intermediate, such as pigs, in which the trachea expresses both human-type and avian-type receptors (Ito et al. 1998Go; Suzuki et al. 2000Go). These phenomena indicate that chickens and quails also play an important role in the evolution of influenza viruses by acting as intermediate hosts, in which avian influenza viruses can be amplified and transmitted to other animal species (Lin et al. 2000Go). Some researchers (Perez, Webby, et al. 2003Go; Wan and Perez 2006Go) have also suggested that land-based birds act as mixing vessels or disseminators of avian/mammalian reassortant influenza A viruses.

Intestinal tracts of quails and chickens are infection and replication sites for most avian influenza viruses (Webster et al. 1978Go; Hinshaw et al. 1980Go). In this study, we investigated the sialyl-Gal sugar chains in the quail and chicken intestine in order to determine the molecular mechanism of receptor specificity and the reassortant from different strains.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Binding of both influenza viruses that preferentially recognize receptors with saccharides terminating in Sia {alpha}2-3Gal (avian-type viruses) and Sia{alpha}2-6Gal (human-type viruses) to epithelial cells in quail and chicken colons
In order to determine the binding specificity of quail and chicken epithelial cells with influenza viruses, we selected two strains, the avian-type strain A/PR/8/34 (H1N1) (PR8) and the human-type strain A/Memphis/1/71 (H3N2) (Mem71), because a thin-layer chromatography (TLC)/virus-binding assay showed that PR8 and Mem71 bound only to an avian-type receptor [Neu5Ac{alpha}2-3Gal-linked sialylparagloboside (SPG)] and only to a human-type receptor (Neu5Ac{alpha}2-6Gal-linked SPG), respectively (Figure 1A). The virus binding to epithelial cells in quail and chicken colons was investigated by an immunohisological fluorescence method. As shown in Figure 1B, both PR8 and Mem71 strains could bind to epithelial cells of both quail and chicken colon villi (transverse sections). There was no viral antigen detected in mock-infected sections. After pretreatment of their tissue sections at 37 °C for 1 h with 100 mU/mL sialidase from Arthrobacter ureafaciens, their binding activities were strongly inhibited, indicating that both quail and chicken colons had some compounds containing Sia residues responsible for the binding of influenza viruses, both PR8 and Mem71 strains.


Figure 1
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  		Fig. 1. Binding of epithelial cells in quail and chicken intestinal (colon) villi by both avian-type (PR8) and human-type (Mem71) influenza A viruses. (A) A TLC/virus-binding assay was carried out to investigate the binding specificities of PR8 and Mem71 strains to SPGs, (Neu5Ac{alpha}2-3Gal- and Neu5Acß2-6Gal-linked SPGs), which are used as standard substrates for the study of influenza A virus receptor-binding specificity (Suzuki et al. 1986Go, 1992). (B) The binding specificities of influenza viruses to epithelial cells in quail and chicken colons were detected with an immunohistological fluorescence method as described in detail in the Materials and methods section. Briefly, the sections fixed on glass quail and chicken colon villi (transverse section) were first treated with an acetate buffer (100 mM, pH 5.5) in the presence (lower group) or absence (upper group) of 100 mU/mL of sialidase from A. ureafaciens (Sigma) at 37 °C for 1 h, and then incubated with influenza viruses at 4 °C for 12 h. The sections were incubated with anti-NP mAb (4E6) and subsequently with FITC-labeled anti-mouse IgG + M antibody. The sections were observed with a laser scanning confocal microscope (LSM510, Zweiss). Bars represent 50 µm.

 
Replication of influenza A viruses in cultured cells from the quail colon
In order to investigate further the replication of influenza A viruses in the animal colon, cells were first isolated from the quail colon by trypsinization with 10 µg/mL acetylated trypsin for 1 h at 37 °C, and then the isolated cells were inoculated with PR8 or Mem71 strain at 0.01 plaque-forming units (PFU). At 12 h after the inoculation, we detected the expression of the viral nucleoprotein in the cytoplasm of quail cells (Figure 2A). Productive viral infection of both PR8 and Mem71 in primary cultured cells isolated from the quail colon was demonstrated by an increase in the viral yield in culture supernatants (Figure 2B).


Figure 2
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  		Fig. 2. Infectivity of influenza A viruses in epithelial cells isolated from quail colon. (A) Expression of viral proteins in epithelial cells isolated from quail colon infected with PR8 or Mem71 strain was detected with an anti-NP mAb (4E6) as described in detail in the Materials and methods section. (B) Replication of the PR8 (red) or Mem71 (blue) strain in the epithelial cells from quail for in different times (at 24, 48, and 72 h postinfection) was determined by a plaque assay. The data are expressed as mean ± SD of three independent experiments. Each experiment was carried out in duplicate (Shinya et al. 2005Go).

 
The results indicated that both avian-type and human-type viruses could infect and replicate in quail colon cells.

Existence of both Sia{alpha}2-3Gal- and Sia{alpha}2-6Gal-terminated sialylglycoconjugates in epithelial cells of quail and chicken colons
The combination of the two lectins Maackia amurensis agglutinin (MAA) (specific for Sia{alpha}2-3Gal) and Sambucus nigra agglutinin (SNA) (specific for Sia{alpha}2-6Gal) enables the type of Sia-Gal linkage on sialylated N- and/or O-linked carbohydrate side chains to be distinguished. In this study, we used these two lectins to investigate the type of Sia-Gal linkage in epithelial cells of quail or/and chicken colons with a lectin detecting assay in situ. As can be seen in Figure 3, epithelial cells of both quail and chicken colons showed a positive reaction with both SNA and MAA; no positive reaction was observed without each lectin (data not shown). After pretreatment of colon tissues at 37 °C for 1 h with 100 mU/mL sialidase from A. ureafaciens, which cleaves {alpha}2-3-, {alpha}2-6-, {alpha}2-8-, and {alpha}2-9-linked Sias of N- and/or O-linked glycans, their binding activities were strongly inhibited as was observed in the virus binding experiment (Figure 3A). This finding showed that both Sia{alpha}2-6Gal and Sia{alpha}2-3Gal linkages exist on epithelial cells in quail and chicken colons. Furthermore, after pretreatment of colon tissues at 37 °C for 6 h with 200 U/mL peptide N-glycosidase F (PNGase F), their binding activities were also inhibited (Figure 3B). This result indicates that host cell N-linked sialoglycoproteins play an important role in infection of influenza A virus, in accordance with results of a study (Chu and Whittaker 2004Go). The staining by each lectin did not completely disappear after PNGase F treatment, and PNGase F could not release sugar chains of O-glycans and glycolipids. These results indicate that the lectins may also bind to O-glycans and glycolipids.


Figure 3
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  		Fig. 3. Existence of both Sia{alpha}2-3Gal and Sia{alpha}2-6Gal linkages in quail and chicken colons detected by lectin staining. The sections fixed on glass were quail and chicken colon pretreated with an 100 mM acetate–HCl buffer (pH 5.5 for sialidase, pH 7.5 for PNGase F) in the presence or absence of sialidase from A. ureafaciens (100 mU/mL) (Sigma) at 37 °C for 1 h (A) or PNGase F (200 U/mL) at 37 °C for 6 h (B). Both DIG-labeled MAA lectin specific for Sia{alpha}2-3Gal and DIG-labeled SNA lectin specific for Sia{alpha}2-6Gal bound to quail and chicken colon epithelium, and then their binding specificities detected with polyclonal sheep anti-DIG Fab fragments conjugated with alkaline phosphatase according to the protocol provided by the manufacturer. The images were observed under a microscope (40x). Bars represent 50 µm.

 
Both glycoprotein and glycolipid fractions from epithelial cells in quail and chicken intestines contain components that bind to avian- and human-type viruses
To determine whether virus-binding components exist in quail and chicken colons, we harvested glycoprotein and glycolipid fractions from quail and chicken colon epithelial cells.

For detecting their glycolipids, acidic lipids of the total lipid fraction were purified with a Phenyl Sepharose CL-4B column chromatography (Waki et al. 1994Go), and then used for TLC/virus-binding (Figure 4A) and lectin-binding detection (Figure 4B). Both avian- and human-type viruses (PR8 and Mem71) and both sialyl linkage-specific lectins (SNA and MAA) bound to several glycolipids. We also used monoclonal antibodies (mAbs) to probe {alpha}2-3GM3 and {alpha}2-3- and {alpha}2-6SPG (Figure 4C). The results indicated that the glycolipids bound with PR8 ({alpha}2-3-specific) were {alpha}2-3GM3 and {alpha}2-3SPG, whereas the glycolipid that bound with Mem71 ({alpha}2-6-specific) was {alpha}2-6SPG. No specific bindings was detected in virus (–), lectin (–), and mAb (–) experiments.


Figure 4
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  		Fig. 4. Characterization of glycolipids from epithelial cells in quail and chicken colon. (A) The binding specificity of influenza A virus (PR8 or Mem71 strains) to glycolipids from epithelial cells of quail and chicken colon was detected with a TLC/virus-binding assay as described in detail in the Materials and methods section. (B) Lectin staining detected the existence of both Sia{alpha}2-3Gal and Sia{alpha}2-6Gal linkages. (C) Immunostaining assay detected {alpha}2-3GM3 and {alpha}2-3- or {alpha}2-6SPG with mAbs. SD, standard, such as {alpha}2-3GM3, {alpha}2-3SPG, and {alpha}2-6SPG; Ck, acidic glycolipids from chicken colon; Qu, acidic glycolipids from quail colon.

 
To detect sialoglycoproteins, cell proteins solubilized with 2% sodium dodecyl sulfate (SDS) were applied to a 10–20% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel to detect the virus- and lectin-binding components (Figure 5). The bands that bound to influenza virus strains PR8 and Mem71, were visualized and their molecular weights were determined to be 60–100 kDa, similar to that of fetuin (Ft) (68 kDa). The glycoprotein bands that bound to SNA and MAA were also detected. These bands disappeared after pretreating the glycoproteins with 100 mU/mL sialidase from A. ureafaciens at 37 °C for 1 h, indicating that the Sias of both glycoproteins and glycolipids in quail and chicken colons were important moieties for binding the avian- and human-type strains. No specific bindings were detected in virus (–) and lectin (–) experiments.


Figure 5
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  		Fig. 5. Detection of the glycoproteins from epithelial cells in quail and chicken colon by virus-binding assay (A) and lectin western blots (B). (A) The binding specificity of influenza virus to glycoproteins from epithelial cells in quail and chicken colon was detected as described in the Materials and methods section. (B) Lectin western blots detected the existence of both Sia{alpha}2-3Gal and Sia{alpha}2-6Gal linkages of the glycoproteins from epithelial cells in quail and chicken colon. The glycoproteins used for this experiment and Ft as a positive control were pretreated with an acetate buffer (100 mM, pH 5.5) in the presence (Sialidase +) (100 mU/mL) or absence (Sialidase–) of sialidase from A. ureafaciens at 37 °C for 1 h. Ck, glycoproteins from chicken colon; Qu, glycoproteins from quail colon.

 
Taken together, the results indicate that sialoglycoproteins and sialo glycolipids that carry Sia{alpha}2-3Gal and Sia{alpha}2-6Gal linkage responsible for the binding of avian and human influenza viruses are present in the pathological sites such as the intestines of the quail and chicken.

Molecular species of Sias and chemical determination of the N-linked sialyl sugar chain structures in epithelial cells of quail and chicken colons
The Sia species were determined by high-performance liquid chromatography (HPLC) using 1,2-diamino-4,5-methylenedioxy-benzene (DMB) as a fluorogenic compound as previously described (Suzuki et al. 1997Go). As shown in Figure 6, the molecular species of Sias in both quail and chicken colons were 5-N-acetylneuraminic acid (Neu5Ac). No N-glycolyneuramic acid (Neu5Gc) acid was detected.


Figure 6
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  		Fig. 6. The Sia species and their linkages in epithelial cells of the quail and chicken colon. The Sia species were determined with a fluorescence HPLC method as described in the Materials and methods section. (A) Standard mixture of Neu5Ac and Neu5Gc; (B) epithelial cells of chicken colon; and (C) epithelial cells of quail colon.

 
We further determined the structures of sialylated N-glycans derived from intestinal epithelial tissues of the chicken and quail. N-Glycans were released from acetone powders from the intestinal epithelial tissues by glycoamidase A digestion and then labeled with 2-aminopyridine. The pyridylamino (PA)–oligosaccharide mixture was applied to a diethylamino ethanol (DEAE) column to separate neutral, monosialyl, and disialyl oligosaccharide fractions. The molar ratios of the neutral, monosialyl, and disialyl fractions were 93.9, 4.3, and 1.8%, respectively, for the chicken and 90.3, 6.3, and 3.4%, respectively, for the quail. The sialylated PA-glycan fractions were further fractionated by an octadecyl silica (ODS) column. Figure 7 shows the elution profiles of mono- and di-sialyl oligosaccharides derived from chicken and quail intestinal epithelial tissues on the ODS column. The main fractions were further separated on an amide–silica column. The isolated PA-glycans were identified by inspection of their elution time data on ODS and amide columns, which were confirmed by cochromatography on the ODS column of the sample PA-glycans with the corresponding reference compound and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analyses. The sialyl N-glycan structures thus determined are shown in Table I. These data indicated that, for both the chicken and quail, bi-antennary complex-type glycans {alpha}2-6 sialylated at the terminal Gal residue(s) are major (more than 79%) sialyl N-glycans expressed by intestinal epithelial tissues.


Figure 7
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  		Fig. 7. The N-glycosyltion profiles on the ODS column of mono- and di-sialyl N-glycans isolated from quail (A and B) and chicken (C and D) intestinal epithelial cells. A and C, monosialyl N-glycans; B and D, disialyl N-glycans. Neutral and mono- and di-sialyl N-glycans from intestinal epithelial cells were separated on the DEAE column in advance. The peaks were corded alphabetically in order of GU (ODS) in the profiles.

 

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  		Table I. The structures and relative quantities of the mono- and di-sialyl PA-oligosaccharides derived from quail and chicken intestinal epithelial cells

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Influenza viruses recognize specifically selected sialyl sugar chains and molecular species of Sia on the host cell membranes. We have demonstrated that influenza A and B viruses can strongly bind to monosialo-lactosamine type I and II, Sia{alpha}2-3(6)Galß1-3(4)GlcNAcß1- (Suzuki, Matsunaga, and Matsumoto 1985Go; Suzuki, Matsunaga, Nagao, et al. 1985Go; Suzuki et al. 1986Go, 1992, 1997; Ito, Suzuki, Mitnaul, et al. 1997Go; Suzuki 2005Go), which are expressed in glycoproteins and gangliosides in the host cell membranes, and we have determined the receptor-binding specificities of human, swine, avian, and equine influenza A viruses and also the sialyl linkages present on cells of the host target organs, trachea epithelium for humans, pigs, and horses and intestinal mucosa for birds, using lectins that recognize Sia–Gal linkages, {alpha}2-3 or {alpha}2-6. Human influenza viruses isolated from Madin–Darby canine kidney cells bind to the Neu5Ac{alpha}2-6Gal sequence, but equine and avian viruses bind predominantly to the Neu5Ac{alpha}2-3Gal sequence (Ito, Suzuki, Takada, et al. 1997Go; Suzuki et al. 2000Go). Swine viruses bind equally to both Neu5Ac{alpha}2-6Gal and Neu5Ac{alpha}2-3Gal or with a slight predominance toward Neu5Ac{alpha}2-6Gal (Suzuki et al. 1992Go; Ito, Suzuki, Takada, et al. 1997Go; Ryan-Poirier et al. 1998Go). The human upper trachea expresses Sia{alpha}2–6Gal (Baum and Paulson 1990Go; Couceiro et al. 1993Go; Shinya et al. 2006Go; Van Riel et al. 2006Go), whereas the horse trachea and duck intestinal mucosa express the {alpha}2-3 linkage. In contrast, the pig trachea expresses both {alpha}2-3 and {alpha}2-6 linkages (Ito, Suzuki, Mitnaul, et al. 1997Go; Ito, Suzuki, Takada, et al. 1997Go; Ito et al. 2000Go; Suzuki et al. 2000Go). We have also demonstrated that Neu5Gc{alpha}2–3Gal recognition is essential for viral replication in ducks (Ito et al. 2000Go) and in horses (Suzuki et al. 2000Go). Due to their ability to support replication of both avian and human influenza viruses, pigs have been implicated as intermediate hosts, serving as mixing vessels for avian and human viruses. It has been reported that some H9N2 influenza viruses isolated from quails in China have already acquired a preference for binding to Neu5Ac{alpha}2-6Gal receptors like human strains (Matrosovich et al. 2001Go; Saito et al. 2004Go), indicating that this virus has a potential for human-to-human transmission. We (Ito et al. 1997Go) previously found that a single amino acid substitution in influenza virus H3 HA resulted in a marked change in receptor-binding specificity. Culturing human influenza A (H3 subtype) viruses which bind to a human-type receptor (Sia{alpha}2-6) in the allantoic membrane of embryonated chicken eggs that have a predominantly Sia{alpha}2-3Gal (bird type) receptor resulted in the generation of a mutated virus that has altered receptor-binding specificity adapted to the bird-type receptor (Sia{alpha}2-3). This change in receptor-binding specificity was accompanied by the appearance of variants in the population with a single amino acid change, Leu-to-Gln at position 226 in the viral HA. These findings suggest that influenza viruses generated in the host target tissue cells may be selected during their replication cycles by the receptor sialosugar chains (Sia{alpha}2-3 or Sia{alpha}2-6) that are present in the host cells, and the selected virus that has a new receptor-binding specificity may be able to cross over the host barrier. These results indicate that bird influenza viruses that recognize the Neu5Ac{alpha}2-3Gal receptor can be mutated in the infected chicken and/or quail body during their circulation in poultry to a human type that binds to the human receptor Neu5Ac{alpha}2-6Gal sialyl sugar chains.

In experimental infections, avian influenza viruses transmitted poorly to primates (Murphy et al. 1982Go; Snyder et al. 1987Go; Beare and Webster 1991Go; Matrosovich et al. 2004Go), and human isolates did not efficiently infect ducks (Webster et al. 1978Go; Kida et al. 1980Go; Hinshaw et al. 1983Go). However, direct transmission of a highly pathogenic avian influenza virus, H5N1, to humans was reported in Hong Kong in 1997 (Claas et al. 1998Go; Subbarao et al. 1998Go) and also in numerous countries in Asia, The Middle East, Europe, and Africa since 2003 (Suzuki 2005Go; Webster et al. 2006Go, Enserink 2006Go, Kuiken et al. 2006Go; World Health Organization, http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_03_01/en/index.html). Recently, it has been reported that influenza viruses enter the human airway epithelium through specific nonciliated cells, whereas avian viruses, as well as an egg-adapted human virus variant with an avian virus-like receptor-binding specificity, mainly infected ciliated cells, and this pattern correlated with the predominant localization of receptors for human viruses ({alpha}2-6-linked Sias) on nonciliated cells and of receptors for avian viruses ({alpha}2-3-linked Sias) on ciliated cells (Matrosovich et al. 2004Go). We have also demonstrated that human trachea primary epithelial cells express both the Sia{alpha}2-6Gal receptor for human influenza viruses and the Sia{alpha}2-3Gal receptor for avian influenza viruses (Kogure et al. 2006Go). Recently, {alpha}2-3-linked Sia was found on human airway nonciliated cuboidal bronchiolar cells at the junction between the respiratory bronchiole and alveolus, i.e., lower respiratory tract (Shinya et al. 2006Go, Van Riel et al. 2006Go). These findings suggest that direct exposure of human trachea epithelial cells to high concentrations of H5N1 can mediate direct infection of the avian virus in humans though sialyl{alpha}2-3Gal receptors in the trachea.

In the present study, we found that epithelial cells of quail and chicken colons contain both Neu5Ac{alpha}2-3Gal- and Neu5Ac{alpha}2-6Gal-terminated sialylglycoproteins and Sialylglycolipids, and we identified 20 kinds of mono- and di-sialyl N-linked oligosaccharides that carry Neu5Ac{alpha}2-3Gal- and/or Neu5Ac{alpha}2-6Gal termini. We also showed that not only an avian-like strain but also a human strain of influenza virus could replicate in primary cultured cells from the quail colon. Our results indicate that the quail and chicken are potential intermediate hosts for avian influenza virus transmission to humans, which may cause a new pandemic for influenza.

All known HA and NA subtypes of influenza A viruses circulate in wild aquatic birds (Webster et al. 1992Go). The H2N2 and H3N2 subtypes implicated in the 1957 and 1968 pandemics were thought to have been generated as a result of reassortment occurring between avian and human influenza viruses in pigs (Scholtissek et al. 1987Go; Schafer et al. 1993Go), since the pig trachea expresses both Sia{alpha}2-6Gal- and Sia{alpha}2-3Gal-terminated sialylglycoconjugates (Ito et al. 1998Go; Suzuki et al. 2000Go) and both Neu5Ac and Neu5Gc species (Ito, Suzuki, Mitnaul, et al. 1997Go). Recently, some studies (Gambaryan et al. 2002Go; Kim et al. 2005Go; Wan and Perez 2006Go) have suggested that cells in the respiratory and intestinal tracts of chickens have different proportions of both human and avian influenza virus receptors. We further confirmed that quails and chickens are potential intermediate hosts by molecular characterization of their receptor. The recent transmission of avian H5N1 influenza viruses to humans might be a result of adaptation of avian viruses in chickens and/or quails to humans. We found that H5N1 isolated from a boy visiting Fujian Province in China bound to both receptors {alpha}2-3 and {alpha}2-6 (Shinya et al. 2005Go), suggesting adaptation of the avian H5N1 isolated from humans. We also detected similar mutations in avian H5N1 isolated from humans in Hanoi, Vietnam (Le et al. 2005Go). Thus, the initial mutation of highly pathogenic avian influenza viruses (H5N1) into viruses that easily transmit among humans will primarily be an adaptation of receptor-binding specificity of the H5N1 virus from {alpha}2-3 (bird type) to {alpha}2-6 (human type). Very recently, we (Yamada et al. 2006Go) have determined amino acids (number 182 and 192) in the highly pathogenic avian H5N1 virus HA that are responsible for the binding to human-type receptors (Sia{alpha}2-6Gal). Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to those that recognize the human receptor. These mutations may act as an initial molecular signals for the next influenza pandemic. Perez, Webby, et al. (2003Go) suggested that land-based birds act as mixing vessels or disseminators of avian/mammalian reassortant influenza A viruses. Our study demonstrated that quails and chickens have molecular characterization as potential intermediate hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential. Therefore, we suggest that chickens and/or quails, as well as pigs need careful supervision for the next expected influenza pandemic. Although highly pathogenic H5N1 viruses replicate preferentially in the respiratory tract of the duck (Sturm-Ramirez et al. 2005Go), mutation of a lower pathogenic virus to a highly pathogenic virus can occur more easily in the digestive tract than in the respiratory tract of wild birds, because the symptom of infection in the respiratory tract are more serious than those in the digestive tract. Virus reassortment usually occurs when the same host cells have been coinfected by two or more strains (Hinshaw et al. 1980Go; Webster et al. 1992Go; Suzuki 2005Go). From these results, we suggest that the digestive tracts of chickens and quails are sites in which new influenza viruses with human pandemic potential may be generated.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Conflict of interest statement
 Acknowledgments
 References
 
Materials
Glycoamidase A (also known as glycopeptidase A, EC3.5.1. 52) from sweet almond and ß-galactosidase from jack beans were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Acetylated trypsin (T-6763), N, N-dimethyl-p-phenylenediamine dihydrochloride (DEPED), 4-chloro-1-naphthol (4-CN), pepsin, pronase, and Ft from fetal bovine serum (F3385), Neu5Gc (G9793), Neu5Ac (A0812), sialidase from A. ureafaciens (N3786), PNGase F (P7365), and GM3 [Neu5Ac{alpha}2-3Galß1-4Glcß1-1'Cer] were purchased from Sigma Chemical Co. (St Louis, MO). SNA lectin, specific for Sia{alpha}2-6Gal/N-acetylgalactosaminide, and MAA lectin, specific for Sia{alpha}2-3Gal were obtained from Boehringer Mannheim Biochemicals (Mannheim, Germany). Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharma., Co., Ltd. (Tokyo, Japan). Fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin-M and -G (IgG + M) antibody was purchased from ICN Pharmaceuticals, Inc. (Aurora, OH). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG + M was obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). A glycan determination kit was purchased from Boehringer Mannheim Biochemicals. Two SPGs [Neu5Ac{alpha}2-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-1' Cer ({alpha}2-3SPG) and Neu5Ac{alpha}2-6Galß1-4GlcNAcß1-3Galß1-4Glcß1-1' Cer ({alpha}2-6SPG)] were used as receptor substrates and prepared from human placenta (Taki et al. 1988Go) and human meconium (Nilsson et al. 1981Go), respectively. mAbs directed to {alpha}2-3SPG and {alpha}2-6SPG were provided by Dr Tai, Sekagaku Corporation.

Preparation of viruses and antibodies
In this study, we used two influenza viruses [PR8, which preferentially binds to avian-type receptor (Sia{alpha}2-3Gal linkage), and Mem71, which preferentially binds to human-type receptor (Sia{alpha}2-6Gal linkage)] (Figure 1A) to probe host cell infections by influenza virus. The two strains were each propagated in the allantoic cavities of 11-day-old chicken eggs at 35 °C for 48 h and then purified by sucrose density gradient centrifugation (Suzuki, Matsunaga, and Matsumoto 1985Go) and frozen at –80 °C. Viral HA units were determined in micrometer plates using 0.5% guinea-pig erythrocytes as described previously (Perez, Lim, et al. 2003Go).

Rabbit anti-influenza virus antibodies, anti-P50 was raised by immunization of A/X31 (H1N2), which is a reassortant between A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains, and anti-Mem was raised by immunization of A/Memphis/1/71 (H3N2) strain. Anti-nucleoprotein (NP) mAb (4E6) was prepared by immunizing A/Memphis/1/71 as antigen.

Animals and tissue preparation
Quails (Coturnix japonica) and chickens were purchased from poultry markets in Hangzhou, China, and in Kanazawa, Japan. Epithelial cells of quail and chicken colons were prepared from this source of animal obtained from the Institute of Bioengineering, Zhejiang Academy of Medical Sciences, Hangzhou, China.

Viral protein expression and virus replication in host cells
For preparation of epithelial cells, mucosa tissues were scratched from the quail colon, and were then trypsinized with 10 µg/mL acetylated trypsin for 1 h at 37 °C and centrifuged at 300g twice at 4 °C. The isolated cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 °C for viral protein expression and virus replication.

Resuspended cells isolated from quail colon epithelial cells (106/mL) were incubated with 0.01 PFU of viruses for 1 h with gentle shaking. After centrifugation at 300g for 5 min to remove unbound virus, the cells were plated in wells of a 96-well plate for detection of viral protein expression and of a 6-well plate for virus replication.

For detection of viral protein expression, the cells in the 96-well plate were fixed with absolute methanol for 5 min at room temperature after incubation for 12 h. After rinsing thoroughly in phosphate-buffered saline (PBS), the fixed cells were incubated with an anti-NP mAb (4E6) for 1 h at room temperature. After three washes with PBS, the cells were incubated with FITC-labeled anti-mouse IgG + M antibody for 1 h at room temperature. The images were observed under a fluorescence microscope.

For the virus replication experiment, replicated virus titer in the culture fluid of each of the infected and uninfected control wells was determined by a plaque assay as described previously (Miyamoto et al. 1998Go) after incubation at 24, 48, and 72 h.

Immunohistological fluorescence microscopy to detect the binding specificity of influenza virus to host tissues
Fresh tissues from quail and chicken colons were immersed overnight in 4% paraformaldehyde (PFA) in PBS at room temperature, and the fixed tissues were then embedded in paraffin wax. Sections (5 µm in thickness) were cut and placed on gelatin-coated glass slides. These sections were used for immunohistological fluorescence staining and lectin detecting assay.

Immunohistological fluorescence staining was used to detect the binding specificity of influenza virus to host tissues. Briefly, tissues sections sliced to 50 µm in thickness were first incubated in 100 mM acetate buffer (pH 5.5) in the presence or absence of 100 mU/mL sialidase from A. ureafaciens (Sigma) at 37 °C for 1 h. The nonspecific reaction was blocked with 1% fatty acid-free bovine serum albumin in PBS. Then the tissues sections were incubated in PBS containing 256 HA U/mL of influenza viruses (PR8 or Mem71 strain) for 12 h at 4 °C. After rinsing off unbound virus with a PBS, the sections were incubated with anti-NP mAb (4E6) and subsequently with an FITC-labeled anti-mouse IgG + M antibody for 2 h at 4 °C. The sections were observed with a laser scanning confocal microscope (LSM510, Zweiss).

Extraction and immunochemical analyses of glycolipids from epithelial cells in quail and chicken colons
Total lipids from epithelial cells in quail and chicken colons were extracted twice in a solvent system of chloroform/methanol (1v : 1 v), and then separated into gangliosides and other lipids using Phenyl Sepharose column chromatography (Phenyl Sepharose CL-4B column, obtained from Amersham Pharmacia, Uppsala, Sweden) as described previously (Waki et al. 1994Go).

Virus binding, immunostaining, and lectin-detecting assay by TLC of acidic glycolipids from quail and chicken colons were performed by the procedure described previously (Suzuki et al. 1992Go). Briefly, after developing with a solvent system of chloroform/methanol/water containing 12 mM MgCl2 (5 : 4 : 1 by vol.), the plastic plate was dried and soaked for 1 h in PBS supplemented with 1% polyvinypyrrolidone (PVP) and 1% egg albumin to block nonspecific virus, antibody or lectin binding. In the virus-binding experiment, the plates were incubated with influenza A viruses (256 HA U/mL) at 4°C overnight. After washing well with PBS, the plates were incubated with polycolonal antibodies against influenza viruses (anti-P50 for PR8 strain and anti-Mem for Mem71 strain). In the experiment using antibodies, the plates were incubated for 1 h at room temperature in PBS containing mouse mAbs against {alpha}2-3GM3, {alpha}2-3SPG, or {alpha}2-6SPG, respectively. These plates were further incubated for 1 h in PBS containing the HRP-conjugated goat anti-mouse IgG + IgM. After washing several times with PBS, the plates were stained with a solution containing 100 mM citrate buffer (pH 6.0), 60 mM DEPED in acetonitrile, 100 mM 4-CN in acetonitrile, and 3% H2O2 (5 : 1 : 1 : 0.005 by vol.) for 15 min at room temperature.

We carried out the lectin detecting experiment according to the protocol provided by the manufacturer (glycan determination kit; Boehringer Mannheim Biochemicals). Briefly, the sections of quail and chicken colons fixed on the glass were pretreated with 100 mM acetate–HCl buffer (pH 5.5 for sialidase, pH 7.5 for PNGase F) in the presence or absence of sialidase from A. ureafaciens (100 mU/mL) at 37 °C for 1 h or PNGase F (200 U/mL) (P7367, Sigma) at 37 °C for 6 h. After rinsing the fixed tissue specimen in PBS (pH 7.4), the plates were blocked with a specific blocking reagent for 1 h at room temperature. After rinsing thoroughly in PBS, the plates were incubated with 50 µL of digoxigenin (DIG)-labeled SNA lectin (1 µg/mL; specific for Sia{alpha}2-6Gal/N-acetylgalactosaminide) or MAA lectin (5 µg/mL; specific for Sia{alpha}2-3Gal) for 1 h at room temperature and with anti-DIG-alkaline phosphatase (anti-DIG-AP). Images were observed under a light microscope (Olympus).

Analyses of glycoproteins from epithelial cells in quail and chicken colons
Western blotting was used to detect the binding activity of the viruses to the glycoprotein from the host cell membrane. Total proteins (50 µg) from epithelial cells of quail and chicken intestines were subjected to SDS–PAGE through a 10– 20% polyacrylamide running gel. Proteins were electrophoretically transferred to immune-blot PVDF membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked for 1 h in PBS containing 3% skim milk and 0.1% Tween-20. Subsequent procedures, including virus binding and lectin detecting assay, were similar to those used in the experiment for analyses of glycolipids.

Fluorometric HPLC analyses of the molecular species of Sia
Fluorometric determination of Neu5Ac and Neu5Gc was conducted by an HPLC method using DMB as previously described (Suzuki et al. 1997Go). The acidic fraction of lipids from epithelial cells of the quail or chicken intestinal tract was hydrolyzed with 0.2 mL of 25 mM sulfuric acid. The hydrolysate was reacted with DMB reagent and heated at 60 °C for 2.5 h in the dark to develop the fluorescence for determination of Sias. A 10 µL aliquot of the resulting solution was applied to an ODS column (TSKgel ODS-80Ts column, obtained from Tosoh Co. Ltd., Tokyo, Japan). The elution times of the individual peaks onto the ODS columns were normalized with reference to Neu5Ac and Neu5Gc. The fluorescence of DMB derivatives was detected at an excitation wavelength of 373 nm and an emission wavelength of 448 nm.

Isolation and characterization of sialylated N-glycans by multi-dimensional mapping
PA derivatives of isomalto-oligosaccharides (4–20 glucose residues) and neutral PA-glycans (code numbers 310.2 and 211.2) were obtained from Seikagaku Kogyo. According to previously described methods, PA-derivatives of sialylated biantennary N-glycans (code numbers 1A1-200.4, 1A2-200.2, 1A2-200.4, 1A1-200.3, 1A1-201.4, 1A1-210.4, 1A2-210.4, 1A1-211.3, 1A1-211.4, 2A1-200.4, 2A1-210.4, 2A1-300.8, 2A1-201.4, and 2A1-211.4) were prepared from human serum glycoproteins, while those of {alpha}2-3-sialylated oligosaccharide (code numbers 1A3-200.4, 1A4-210.4, and 2A3-200.4A) were prepared by using an {alpha}2,3-trans sialidase from Typanosoma cruzi (Nakagawa et al. 1995Go; Takahashi et al. 1995Go). Acetone powdered samples (each 20 mg) of chicken and quail intestinal epithelial tissues were used as starting materials. Structural determination of N-glycans was performed by an HPLC mapping method described previously (Nakagawa et al. 1995Go; Takahashi et al. 1995Go), with slight modifications of the preparation of PA derivatives of the N-glycans. Briefly, the samples were treated with pepsin plus glycoamidase A in 0.1 M citrate–phosphate buffer, pH 4.0, at 37 °C overnight. The resultant peptides were further digested by pronase in 1 M Tris-HCl buffer, pH 8.0, at 37 °C overnight. The released N-glycans were collected by a BIO-GEL P-2 gel column (Bio-Rad) and then reductively aminated with 2-aminopyridine by the use of sodium cyanoborohydride (Yamamoto et al. 1989Go). The PA-glycans were applied to a DEAE column to fractionate the sialylated oligosaccharides according to their anionic charges. The sialyl PA-oligosaccharides were applied to an ODS column, and then each separated fraction was further applied to an amide-silica column. The elution times of the individual peaks on the ODS and amide-silica columns were normalized with reference to the PA-derivatized isomalto-oligosaccharides of polymerization degree 4–20 and represented by glucose units (GUs). Thus, a given compound from these two columns provided a unique set of GU values, which corresponded to coordinates of the two-dimensional HPLC map. The PA-oligosaccharides were identified by comparison with the HPLC data of approximately 500 reference PA-oligosaccharides in a homemade web application, GALAXY (http://www.glycoanalysis.info/) (Takahashi and Kato 2003Go). The PA-oligosaccharides were cochromatographed with reference PA-oligosaccharides on the columns to confirm their identities.

MALDI-TOF-MS analysis
PA-oligosaccharides were subjected to MALDI-TOF-MS analysis. MALDI-TOF-MS data were acquired in the positive and negative modes using AXIMA-CFR (Shimadzu) operated in the linear mode. The matrix solution was prepared as follows: DHB (10 mg) was dissolved in 1 : 1 (v/v) of acetonitrile/water (1 mL). Stock solutions of PA-glycans were prepared by dissolving them in pure water. One microliter of sample solution was mixed on the target spot of a plate with 1 µL of matrix solution and then allowed to dry in air.


   Conflict of interest statement
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
Acknowledgments
 References
 
None declared.


   Acknowledgments
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
References
 
A part of this work was supported by a grant of the JSPS Postdoctoral Fellowship for Foreign Researchers (15·03131) from the Japan Society for the Promotion of Science and by grant-in-aid (17046017 and 17390022) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a grant from Zhejiang Natural Science Fund and Zhejiang Science and Technology, China. A part of this work was also supported by Mitsubishi Foundation and Heiwa Nakajima Foundation.


   Abbreviations
 
4-CN, 4-chloro-1-naphthol; DEAE, diethylamino ethanol; DEPED, N, N-dimethyl-p-phenylenediamine dihydrochloride; DIG, digoxigenin; DMB, 1,2-diamino-4,5-methylenedioxy-benzene; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; Ft, fetuin; Gal, galactose; Gus, glucose units; HA, hemagglutinin; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; IgM and IgG, immunoglobulin-M and -G; MAA, Maackia amurensis agglutinin; mAbs, monoclonal antibodies; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Mem71, A/Memphis/1/71 (H3N2); Neu5Ac, 5-N-acetylneuraminic acid; Neu5Gc, 5-N-glycolylneuraminic acid; ODS, octadecyl silica; PA, pyridylamino; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PFU, plaque-forming units; PNGase F, peptide N-glycosidase F; PR8, A/PR/8/34 (H1N1); PVP, polyvinypyrrolidone; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Sia, sialic acid; SNA, Sambucus nigra agglutinin; SPG, sialylparagloboside; TLC, thin-layer chromatography.


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
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