Decoding the Distribution of Glycan Receptors for Human-Adapted Influenza A Viruses in Ferret Respiratory Tract

Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Department of Biological Engineering, Cambridge, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2012; 7(2):e27517. DOI: 10.1371/journal.pone.0027517
Source: PubMed


Ferrets are widely used as animal models for studying influenza A viral pathogenesis and transmissibility. Human-adapted influenza A viruses primarily target the upper respiratory tract in humans (infection of the lower respiratory tract is observed less frequently), while in ferrets, upon intranasal inoculation both upper and lower respiratory tract are targeted. Viral tropism is governed by distribution of complex sialylated glycan receptors in various cells/tissues of the host that are specifically recognized by influenza A virus hemagglutinin (HA), a glycoprotein on viral surface. It is generally known that upper respiratory tract of humans and ferrets predominantly express α2→6 sialylated glycan receptors. However much less is known about the fine structure of these glycan receptors and their distribution in different regions of the ferret respiratory tract. In this study, we characterize distribution of glycan receptors going beyond terminal sialic acid linkage in the cranial and caudal regions of the ferret trachea (upper respiratory tract) and lung hilar region (lower respiratory tract) by multiplexing use of various plant lectins and human-adapted HAs to stain these tissue sections. Our findings show that the sialylated glycan receptors recognized by human-adapted HAs are predominantly distributed in submucosal gland of lung hilar region as a part of O-linked glycans. Our study has implications in understanding influenza A viral pathogenesis in ferrets and also in employing ferrets as animal models for developing therapeutic strategies against influenza.

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Available from: Akila Jayaraman, Oct 03, 2015
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    • "This could enable preliminary assessment of pandemic potential of newly emerged influenza strains by providing an estimation of their affinity for human sialic acid receptors and their ability to be neutralized by existing anti-HA antibodies. Use of our structural model of Hangzhou-H7 for docking analysis predicted that it had a similar affinity for the human α2,6 sialyl-lactose receptor (−6.9 kcal/mol) than the avian α2,3 sialyl-lactose receptor (−6.6 kcal/mol), which could suggest increased pandemic potential [21], [47]. When assessed using the same model, the HA of the high pathogenicity avian H7N7 strain showed a higher affinity for the avian than the human receptor, as might be expected of a non-mammalian adapted strain. "
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    ABSTRACT: The unpredictable nature of pandemic influenza and difficulties in early prediction of pandemic potential of new isolates present a major challenge for health planners. Vaccine manufacturers, in particular, are reluctant to commit resources to development of a new vaccine until after a pandemic is declared. We hypothesized that a structural bioinformatics approach utilising homology-based molecular modelling and docking approaches would assist prediction of pandemic potential of new influenza strains alongside more traditional laboratory and sequence-based methods. The newly emerged Chinese A/Hangzhou/1/2013 (H7N9) influenza virus provided a real-life opportunity to test this hypothesis. We used sequence data and a homology-based approach to construct a 3D-structural model of H7-Hangzhou hemagglutinin (HA) protein. This model was then used to perform docking to human and avian sialic acid receptors to assess respective binding affinities. The model was also used to perform docking simulations with known neutralizing antibodies to assess their ability to neutralize the newly emerged virus. The model predicted H7N9 could bind to human sialic acid receptors thereby indicating pandemic potential. The model also confirmed that existing antibodies against the HA head region are unable to neutralise H7N9 whereas antibodies, e.g. Cr9114, targeting the HA stalk region should bind with high affinity to H7N9. This indicates that existing stalk antibodies initially raised against H5N1 or other influenza A viruses could be therapeutically beneficial in prevention and/or treatment of H7N9 infections. The subsequent publication of the H7N9 HA crystal structure confirmed the accuracy of our in-silico structural model. Antibody docking studies performed using the H7N9 HA crystal structure supported the model's prediction that existing stalk antibodies could cross-neutralise the H7N9 virus. This study demonstrates the value of using in-silico structural modelling approaches to complement physical studies in characterization of new influenza viruses.
    PLoS ONE 07/2014; 9(7):e102618. DOI:10.1371/journal.pone.0102618 · 3.23 Impact Factor
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    • "The distribution of terminal Sias in α2-6 and in α2-3 linkages varies along the respiratory tract, and changes with age and developmental stage [19,23]. Human respiratory tract sialylation patterns have been extensively studied on paraffin embedded tissues, which are lacking much of the secreted mucus layer [23,24]. Here we examine glycosylation and IAV binding to frozen human trachea/bronchus tissues that were frozen and embedded in optimal cutting temperature (OCT) compound. "
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    ABSTRACT: Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established. We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity. IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM. IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.
    Virology Journal 11/2013; 10(1):321. DOI:10.1186/1743-422X-10-321 · 2.18 Impact Factor
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    • "The transmissibility of human-adapted pandemic influenza strains, 1918 H1N1, 1957 H2N2 and 2009 H1N1, in ferrets has been demonstrated to correlate with the specificity and quantitative affinity of the viral surface glycoprotein hemagglutinin (HA) binding to α2→6 sialylated glycans (or human receptors) [7]–[9]. These human receptors are predominantly expressed in the respiratory tract epithelium of humans and ferrets [10]–[12]. The HA of influenza viruses isolated from avian species typically binds to α2→3 sialylated glycans (or avian receptors) [11]. "
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    ABSTRACT: Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.
    PLoS ONE 04/2013; 8(4):e59550. DOI:10.1371/journal.pone.0059550 · 3.23 Impact Factor
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