Avian and human influenza virus compatible sialic acid receptors in little brown bats

Abstract

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.

Figure 1

Co-expression of SA α2,3-Gal and SA α2,6-Gal receptors in little brown bat respiratory tract. Composite confocal images show co-expression of both SA α2,3-Gal (red) and SA α2,6-Gal (green) influenza receptors on the (A) trachea and (B) lung of the little brown bat (LBB). The SA α2,3-Gal receptor is the predominant receptor type on the (A’) respiratory mucosal epithelium of the trachea, while the SA α2,6-Gal receptor type is predominant in the lamina propria and submucosa of the trachea and lungs. (C) Schematic representation of the SA receptor distribution in the respiratory system of LBB shows predominance of SA α2,3-Gal receptor type in the upper respiratory tract, which gradually decreases towards the lower respiratory tract. In contrast, SA α2,6-Gal receptors gradually increases towards the lower respiratory tract. Tissue sections were stained with biotinylated MAAII (red - specific for avian type receptor, SA α2,3-Gal) and FITC labelled SNA (green - specific for human type receptor, SA α2,6-Gal) lectins, and DAPI nuclear stain (blue). A”: haematoxylin and eosin (H and E) stained tracheal tissue section. 1. respiratory epithelium, 2. lamina propria, 3. submucosa, 4. hyaline cartilage B”: H and E stained lung tissue section. 5. alveolar duct, 6. visceral pleura, 7. pulmonary blood capillary.

Figure 2

Co-expression of SA α2,3-Gal and SAα2,6-Gal receptors in LBB digestive tract. Composite confocal images show co-expression of both SA α2,3-Gal (red) and SA α2,6-Gal (green) influenza receptors on the mucosal lining of (A) stomach and (B) intestine of the little brown bat (LBB). The SA α2,3-Gal receptor is the predominant receptor type on the mucosal lining of the (A’) stomach and (B’), intestinal villi SA α2,6-Gal receptor type is more prominent in the lamina propria, muscularis and serosa of the (A) stomach and (B) intestine. Tissue sections were stained with biotinylated MAAII (red - specific for avian type receptor, SA α2,3-Gal) and FITC labelled SNA (green - specific for human type receptor, SAα2,6-Gal) lectins, and DAPI nuclear stain (blue). A”: h aematoxylin and eosin (H and E) stained stomach tissue section. 1. mucosa 2. submucosa 3. mucosal glands. B”: H and E stained intestinal tissue section 4. villus, 5. goblet cell, 6. lamina propria, 7. simple columnar epithelial cell layer, 8. submucosa 9. muscularis.

Figure 3

Widespread binding of avian H5N2 virus to LBB tissues. The confocal images show extensive binding of avian H5N2 virus (A/H5N2/chicken/Pennsylvania/7659/1985) to little brown bat (LBB) (A) trachea, (C), lung and (E) intestine. The virus binding pattern reflects the relative abundance of the SA α2,3-Gal receptors in the trachea, lung and intestine of the little brown bat. The respiratory epithelial cells of trachea and luminal mucosal cells of the intestine show preferential binding to the influenza virus. The lung demonstrates partial virus binding as shown by Cy-5 labeled secondary antibody. (B, D and F) Mock treated trachea, lung and intestine respectively.

Figure 4

Widespread binding of human pandemic H1N1 virus to LBB tissues. Confocal images show extensive binding of human H1N1 virus (A/H1N1/Virginia/2009) to little brown bat (LBB) (A) trachea, (C), lung and (E) intestine. Binding of human pandemic H1N1 virus was in accordance with the abundance of SA α2,6-Gal receptors in these tissues. Virus binding was observed predominantly in the submucosa and the hyaline cartilage of the (A) trachea, alveolar epithelial cells of the (C) lung and to lamina propria, muscularis and serosa of the (E) intestine. (B, D and F) mock treated trachea, lung and intestine respectively.

Figure 5

Scanning electron micrograph showing virus binding. Scanning electron micrograph showing binding of (A) avian H5N2 virus (A/H5N2/chicken/Pennsylvania/7659/1985) and (B) human H1N1 virus (A/H1N1/Virginia/2009) to little brown bat (LBB) trachea. (C) Mock controls were performed omitting virus incubation. Extensive binding of avian H5N2 and human H1N1 viruses to LBB trachea was observed. Virus or mock treated sections were fixed in ethanol and were coated with gold before imaging by a field emission SEM (FE-SEM, Merlin Zeiss) under 5 eV. Virus particles were pseudo-colored using Adobe Photoshop CC.