Comparative distribution of human and avian type sialic acid influenza receptors in the pig

Abstract

Background: A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAalpha2,3-GalG(1-3)GalNAc and SAalpha2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively.

Results: Both SAalpha2,3-Gal and SAalpha2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors from duodenum to colon in the pig.

Conclusions: The extensive presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.

Figure 1

Differential expression of SAα2,6-Gal (SNA lectin) and SAα2,3-Gal (MAA II lectin) receptors in the porcine respiratory tract. Composite confocal images show distribution of SAα2,6-Gal receptors (green) and SAα2,3-Gal receptors (red) with nuclear staining (blue). Representative results that show SAα2,6-Gal receptor as the dominant receptor type on the epithelium of trachea (A), bronchus (B) and bronchiole (C), where epithelial cells and goblet cells are the main contributing cell types. SAα2,3-Gal receptor, on the other hand, is the major receptor in the corresponding sub-epithelial (mucosal) region with sparse concentration of SAα2,6-Gal receptor at blood vessels and mucous/serous glands. Both receptor types are similarly expressed on alveolar lining (D). The specificity of lectin binding is demonstrated on serial tracheal sections stained with haematoxylin and eosin (E), and with both SNA and MAA II lectins on section previously treated with sialidase A, where only faint background binding is detected (F). 1. goblet cell, 2. epithelial lining, 3. gland with occasional blood vessel, 4. submucosal gland, 5. mucosa, 6. smooth muscle, 7. blood vessel. Scale bar = 75 μm.

Figure 2

Discriminating two types of SA α2,3-Gal receptors in bronchiole (A) and colon (B) by FITC labelled MAA I (green), and biotinylated MAA II (red) receptors. In bronchiole, MAA I and MAA II typically show similar binding intensity, with prominent presence of MAA I on the epithelial lining (Aii). In colon, MAA II binding, mainly localised to goblet cells, dominates MAA I; MAA I binding is seen as a fine line bordering the epithelium (Bii). 1. epithelial lining, 2. mucosa, 3. smooth muscle, 4. goblet cell. Scale bar = 75 μm.

Figure 3

Differential expression of SAα2,6-Gal (SNA lectin) and SAα2,3-Gal (MAA II lectin) receptors in the porcine intestinal tract. Composite confocal images show distribution of SAα2,6-Gal receptors (green) and SAα2,3-Gal receptors (red) with nuclear staining (blue). Representative results that show the spatial distribution of both receptor types in the duodenum (A) and colon (B, C). In duodenum, SAα2,6-Gal receptor is more abundant than SAα2,3-Gal receptor concentrated in goblet cells and along the epithelial lining (A). In colon, strong co-expression of SAα2,6-Gal and SAα2,3-Gal receptors is detected in goblet cells and on epithelial lining. Colon goblet cells at the crypts show a higher concentration of SAα2,6-Gal receptor (Cii) than those located towards the luminal surface (Bii). 1. epithelial lining, 2. goblet cell. Scale bar = 75 μm.

Figure 4

Extensive presence of SAα2,6-Gal (SNA lectin) and SAα2,3-Gal (MAA II lectin) receptors in the major porcine organs examined. Composite confocal images, along with corresponding haematoxylin and eosin tissue sections (with the exception of skeletal muscle) for orientation, show distribution of SAα2,6-Gal receptors (green) and SAα2,3-Gal receptors (red) with nuclear staining (blue). Cross section and longitudinal section of skeletal muscle are shown. 1. hepatic sinusoid, 2. portal triad, 3. meninx, 4. neuron, 5. white pulp, 6. red pulp, 7. capsule, 8. glomerulus, 9. renal tubule, 10. capillary, 11. basement membrane.

Figure 5

Virus binding assays with swine H1N1 and avian H2N3 viruses on porcine alveolar and tracheal serial sections are consistent with virus affinity for particular host receptor type. The presence of both SAα2,6-Gal and SAα2,3-Gal receptors in alveoli is mirrored by a similar overlapping binding pattern of swine and avian viruses to pneumocytes. By contrast, the dominant SAα(2,6)-Gal receptor type on tracheal epithelium shows preferential binding of swine H1N1 virus. Control sections are without virus treatment; low level of auto-fluorescence was detected on alveolar section. 1. alveolar pneumocyte, 2. epithelial lining. Scale bar = 50 μm.

Figure 6

Schematic representation of the distribution trend of SAα2,6-Gal (SNA), SAα2,3-Galβ(1-4)GlcNAc (MAA I) and SAα2,3-Galβ(1-3)GalNAc (MAA II) receptors along the porcine respiratory tract. Diagram depicts a qualitative, not quantitative, assessment of receptor presence. Along the epithelial tract, SAα2,6-Gal receptor is dominant, with increasing MAA II lectin binding towards the alveolar region. In the sub-epithelial region, MAA II lectin binding is dominant. MAA I lectin binding is localised to the lower tract.