Ferrets have been used extensively as a model to study influenza A virus transmission and pathogenesis. Upon intranasal inoculation of the virus, ferrets exhibit similar clinical manifestation as that of humans although there seems to be a difference in viral tropism between ferrets and humans. Upper respiratory tract is the primary site of influenza A infection in humans. Apart from upper respiratory tract, involvement of lower respiratory tract (lung hilar region) is also reported in ferrets. Viral tropism is determined by distribution of influenza A glycan receptors, which are recognized by the viral hemagglutinin (HA) during infection.
Plant lectins such as SNA-I have been used to stain ferret tracheal tissues in the past and it is generally known that these tissues predominantly express α2→6 sialylated glycan receptors similar to human tracheal tissues 
. Although the overall distribution of α2–6 glycans is has been studied in ferret respiratory tract, not much is known about the finer structural details of glycan receptors going beyond the sialic acid linkage. Further in order to understand viral tropism, in vitro
binding assay to determine the pattern of viral attachment (PVA) of fluorescein labeled human and avian influenza A viral strains to tissues (human and animal) has been performed 
. Binding of virus was detected by routine immunohistochemical techniques. Although these studies have provided insights into viral binding to specific cell types, they do not fully capture the subtleties of host viral interactions especially in the context of glycan receptor distribution in different cell types 
. Moreover without the proper understanding of the glycan receptor binding specificites of influenza A viruses (going beyond α2–3/α2–6 linkage specificity) and the glycan receptor distribution in the tissues, it is challenging to extrapolate PVA of a particular viral subtype to its tropism.
Recently, with the emergence of glycan array technology, the glycan binding specificities of several plant lectins and influenza A virus hemagglutinins have been extensively characterized. In this study we used a combination of both lectins and recombinant human-adapted HA to systematically stain both the upper and lower respiratory tissues (going all the way to lung hilar region) in ferrets. The use of recombinant human-adapted HAs and co-staining with multiple lectins permitted us to define glycan receptors for human-adapted influenza A viruses going beyond terminal sialic acid linkage and map their distribution parts of the ferret respiratory tract ().
Glycan receptor distribution in ferret respiratory tract.
Our observations support the notion that the receptors for human-adapted influenza A viruses in ferrets are O-linked α2→6 sialylated glycans (based on SNA-I/Jacalin co-staining) that are predominantly distributed in the submucosal glands of the lower respiratory tract (lung hilar region). This notion is also consistent with earlier reports of viral antigens being predominantly found in the submucosal glands in ferret trachea and lung hilar region upon infection with human influenza A virus 
. This is further corroborated by studies involving the use of recombinant HA to stain human tracheal tissue sections. One of the hallmarks of the human-adapted HA (from influenza A viruses which have caused pandemic and seasonal outbreaks) is their predominant binding to the non-ciliated goblet cells. Even in the cultures of differentiated human airway epithelial cells, the human influenza A viruses are found to predominantly infect non-ciliated cells as compared to avian influenza A viruses which target the ciliated cells 
. By multiplexing lectin staining with Sialidase A
treatment, our results suggest that goblet cell region in human tracheal tissue might have a more predominant expression of the sialyl-Tn-antigen motif than the submucosal region in the ferret lung hilum although both these regions appear to be target sites for binding by human-adapted influenza viruses. This result is also supported by recent findings that show increased expression of sialyl-Tn antigen after virus infection due to goblet cell and/or acinar gland neoplasia in ferrets 
It is interesting to note that though glycan receptors for human influenza A viruses are differentially distributed in humans and ferrets, they are predominantly expressed in the context of O-linked glycans in either goblet cells (in humans) or in submucosal glands (in ferrets). Given that these O-linked glycans are predominantly expressed in the context of mucins, our study provides a basis to further investigate the role of mucins in influenza A infection. We speculate that by infecting the mucin secreting cells such as goblet cells and submucosal glands, human-adapted influenza A viruses can be easily encapsulated into respiratory droplets formed during sneezing that can in turn facilitate efficient airborne transmission of the virus. In fact, the efficient transmission via respiratory droplets is a hallmark property of human-adapted viruses in the ferret animal models 
In summary, using a panel of lectins, we have systematically characterized the glycan receptor distribution for influenza A HA in ferret upper and lower respiratory tract, especially receptors relevant to human adapted viruses (). This is needed to understand viral pathogenesis in ferrets in order to truly correlate it with that in humans. Although viral pathogenesis is mediated by a concerted function of other viral proteins and host factors, the distribution of host glycan receptors contributes to viral tropism. Moreover, it is important to have a thorough understanding of glycan receptor distribution for improving anti-influenza drug delivery strategies for especially those drugs, which target glycan receptors such as DAS181, a sialidase fusion protein that cleaves off the sialic acid and hence prevents viral entry