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. 2015 Oct 1;526(7571):122-5.
doi: 10.1038/nature15379. Epub 2015 Sep 23.

The Soft Palate Is an Important Site of Adaptation for Transmissible Influenza Viruses

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Free PMC article

The Soft Palate Is an Important Site of Adaptation for Transmissible Influenza Viruses

Seema S Lakdawala et al. Nature. .
Free PMC article

Abstract

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

Figures

Extended Data Fig. 1
Extended Data Fig. 1. Amino acids in the receptor binding site of H1N1pdm HA that bind to α2,3 and α2,6 glycans
Ribbon diagrams of the 2009 H1N1pdm HA receptor binding pocket interacting with an α2,6 sialic acid in the pocket (a), an α2,3 H1N1pdm HA with α2,3 glycan (b), or α2,3 G222D revertant H1N1pdm HA and α2,6 sialic acid (c).
Extended Data Fig. 2
Extended Data Fig. 2. Replication of α2,3 H1N1pdm virus in ferret respiratory tract
We confirmed that the α2,3 H1N1pdm virus replicated to high titers on days 1, 3, and 5 in different parts of the ferret respiratory tract. Each tissue homogenate is highlighted with a dashed-circle, the gray circles represent washes. Each point represents a single animal. The horizontal black line indicates the mean viral titer on a given day.
Extended Data Fig. 3
Extended Data Fig. 3. Stability of engineered mutations in viruses replicating in the soft palate
Deep sequencing of the HA gene segment from virus populations in the soft palate from 1, 3, 5, and 7 DPI reveals a rapid change at position 222, but no change in the other engineered sites. The engineered sites are highlighted in blue, while the wild-type nucleotide is in orange. Each bar represents a single animal.
Extended Data Fig. 4
Extended Data Fig. 4. Airborne transmission of α2,3 H1N1pdm virus after 48 hour exposure time
One ferret in each pair was infected with 106 TCID50 of the indicated virus, a naïve ferret (referred to as airborne-contact or AC) was introduced into the adjacent compartment 24 hours later. The AC animal was removed from the transmission cage on day 3 post-infection as indicated by the black arrow. Nasal secretions were collected every other day for 14 days. Viral titers from the nasal secretions are graphed for each infected or AC animal. The gray shading indicates the exposure time between the infected and AC animals.
Extended Data Fig. 5
Extended Data Fig. 5. Influenza receptor distribution on ferret soft palate
Hematoxylin and eosin (H&E) staining of the soft palate from an uninfected ferret highlights the nasopharyngeal and oral surfaces. Scale bar is 1.25mm. (a) Areas highlighted in parts b-g are marked with dashed line shapes: square – nasopharyngeal surface (b and e), circle - submucosal gland (d and g) and triangle - oral surface (c and f). H&E staining of these regions, reproduced from Figure 4A-C in the main text, are shown in b-d. Staining with plant lectins specific for α2,6 SA (SNA) and α2,3 SA (MAL II) are shown in e-g. Scale bars are 100μm in images b-g.
Extended Data Fig. 6
Extended Data Fig. 6. SC18 staining of ferret respiratory tissues
Sections of ferret trachea (a) lung (b), soft palate (c and d), biopsy of nasal turbinate tissue with respiratory epithelium (RE) (f) and olfactory epithelium (OLF) (g) were stained with purified SC18 HA protein to identify areas expressing long-chain α2,6 SA. Illustration of ferret head (sectioned along the midline) highlighting the anatomical locations of RE and OLF tissues is depicted in (h). Goblet cells on the respiratory epithelium of the soft palate (nasopharyngeal surface) also stained positive for SC18 (d). Absence of SC18 staining after sialidase A treatment (e) indicates the high specificity of SC18 for the respiratory epithelium of the soft palate. All scale bars are 100μm unless indicated.
Extended Data Fig. 7
Extended Data Fig. 7. Influenza receptor distribution on pig and human soft palate
Pig (a-c) and human (g-i) soft palate tissues were stained with plant lectins SNA and MALII which are commonly used as markers for α2,6 and α2,3 sialic acid respectively. Sialidase A treated control was run for each sample to ensure specificity of plant lectins and are displayed in panels (d-f and j-l). Expression of α2,6 sialic acids (SNA staining) is found on the ciliated respiratory epithelium and goblet cells of the nasopharyngeal surface and in the submucoasl glands of both the pig and human soft palate. Expression of α2,3 sialic acids is low in the pig soft palate and found primarily in goblet cells and submucosal glands. In the human soft palate, MALII (α2,3 sialic acids) staining sensitive to sialidase A treatment is found in the goblet cells and respiratory epithelium of the nasopharyngeal surface and in the basal cells of the oral surface. MALII staining in the submucosal glands was not sensitive to sialidase A treatment. Scale bars are 100μm in all images.
Extended Data Fig. 8
Extended Data Fig. 8. Pathology of the soft palate during infection with α2,3 H1N1pdm
The soft palate was removed from 3 ferrets infected with α2,3 pH1N1 virus on 7 DPI. The tissue sections were stained with hematoxylin and eosin. Black arrows indicate the ciliated respiratory epithelium of the soft palate tissue (nasopharyngeal surface). Scale bars are 100μm in all images.
Extended Data Fig. 9
Extended Data Fig. 9. Quasispecies in putative lysine fence
Deep sequencing analysis of the α2,3 H1N1pdm inoculum revealed a mixed population at 4 lysine residues surrounding the receptor binding site of the HA protein. The lysine fence was restored in viruses from the nasal wash of AC animals from 6 days post-exposure (DPE). Each bar represents a single animal, and each amino acid (aa) that contained a quasispecies is indicated.
Extended Data Fig. 10
Extended Data Fig. 10. Quasispecies of lysine fence in various ferret respiratory tissue sections
Deep sequencing of viruses from respiratory tissues of ferrets infected with α2,3 H1N1pdm. Viruses populations from the soft palate (a), nasal wash (b), nasal turbinates (c), trachea (d), bronchoalveolar lavage (BAL) (e), or lung sections (f) were analyzed and the proportion of lysine, glutamic acid, or asparagine are presented. Each bar represents a single animal. The lung section is an average of the right middle lung lobe and a portion of the left caudal lung tissue.
Fig. 1
Fig. 1. Airborne transmission of receptor specific H1N1pdm viruses
Transmission studies were performed in double secure cages with perforated dividers. One ferret in each pair was infected with 106 TCID50 of the indicated virus, a naïve ferret (referred to as airborne-contact or AC) was introduced into the adjacent compartment 24 hours later. Nasal secretions were collected every other day for 14 days. Viral titers from the nasal secretions are graphed for each infected or AC animal. Transmission of α2,6 H1N1pdm (a) and α2,3 H1N1pdm (b) viruses was similar. The red arrow indicates the peak day of viral shedding for AC animals.
Fig. 2
Fig. 2. Characterization of transmissible α2,3 H1N1pdm viruses
Deep sequencing of the α2,3 H1N1pdm inoculum, nasal wash (NW) from an infected ferret on 1, 3, and 5 DPI, and NW from one AC animal on 6 days post-exposure (DPE), representative of the 3 AC animals, revealed a reversion at residue 222 from G to D (a). Graphical representation of the proportion of reads at each engineered nucleotide is shown. Blue shading represents the α2,3 engineered nucleotide and orange represent the WT nucleotide residue. All other engineered nucleotides were maintained. A G222D reversion, in the context of the other engineered mutations, affects the glycan specificity of the α2,3 H1N1pdm virus (b). The glycans are indicated in the figure legend, orange colors represent α2,6SA and blue colors represent α2,3SA. H1 numbering is used for all amino acid positions.
Fig. 3
Fig. 3. Emergence of the α2,3 G222D H1N1pdm virus in the ferret respiratory tract
Different samples from the ferret respiratory tract: NW (a), respiratory epithelium (RE) of nasal turbinates (NT) (b), soft palate (c), trachea (d), bronchoalveolar lavage (BAL) (e), and combined right middle and left cranial lung sections (f) were collected on 1, 3, 5 and 7 DPI. The RE region of the NT is depicted in Extended Data Fig. 6h. The HA gene from virus populations in these samples were deep sequenced and the proportion of reads with D at position 222 is shown in orange, and G is shown in blue. Each bar represents a single animal. The standard error between the right and left lung sections is shown in f.
Fig. 4
Fig. 4. Comparison of long-chain α2,6 SA expression in the soft palate of ferrets, pigs and humans
H&E staining of the soft palate from an uninfected ferret (a-c), pig (g-i) and human (m-o) highlights the nasopharyngeal, submucosal glands (SMG), and oral surfaces. Purified SC18 HA was used to define long-chain α2,6 SA in these sections from an uninfected ferret (d-f), pig (j-l) and human (p-r). Staining of the nasopharyngeal surface is depicted for each species across the first row, SMG in the second row and oral surface on the last row. A sialidase A treated control was run for each sample to ensure specificity of SC18 HA (not shown). Scale bars are 100μm in all images. Asterisks (*) highlight SC18 positive goblet cells and white arrowheads indicate SC18 positive plasma cells in human soft palate.

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