The soft palate is an important site of adaptation for transmissible influenza viruses (original) (raw)

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The sequences detailed in this manuscript can be found in GenBank under accession numbers CY184674–CY185309.

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Acknowledgements

This research was supported in part by the Intramural Research Program of NIAID, NIH and with federal funds from NIAID, NIH, DHHS under contract number HHSN272200900007C and NIAID/NIH Genomic Centers for Infectious Diseases (GCID) program (U19-AI-110819). This manuscript was reviewed by the NIH’s Intramural Research Program’s Committee on Dual Use Research of Concern (DURC), who concluded that the methods and results do not meet DURC criteria. We thank the NIAID Comparative Medical Branch for technical assistance, Subbarao laboratory members for critical input, N. B. Fedorova from JCVI for technical help, X. J. Meng (Virginia Tech College of Veterinary Medicine) and P. Pineyro (Iowa State University) for pig soft palate tissues, and the Consortium for Functional Glycomics for providing glycans for the glycan array analysis. The data for this manuscript and its preparation were generated while D.E.W. was employed at JCVI. The opinions expressed in this article are the authors’ own and do not reflect the views of the Centers for Disease Control, the Department of Health and Human Services, or the United States government. A.J. and R.S. are supported in part by NIH Merit Award (R37 GM057073-13), National Research Foundation supported Interdisciplinary Research group in Infectious Diseases of SMART (Singapore MIT alliance for Research and Technology) and the Skolkovo Foundation supported Infectious Diseases Center at MIT.

Author information

Author notes

  1. Seema S. Lakdawala, Marlene Orandle & David E. Wentworth
    Present address: †Present addresses: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15217, USA (S.S.L.); National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia, 26505 USA (M.O.); Virology Surveillance and Diagnosis Branch, Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia 30329, USA (D.E.W.).,

Authors and Affiliations

  1. Laboratory of infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA
    Seema S. Lakdawala, Elaine W. Lamirande, Angela R. Shih, Christopher T. Hanson, Leatrice Vogel, Myeisha Paskel & Kanta Subbarao
  2. Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Singapore-MIT Alliance for Research and Technology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA
    Akila Jayaraman & Ram Sasisekharan
  3. J. Craig Venter Institute, Rockville, 20850, Maryland, USA
    Rebecca A. Halpin, Timothy B. Stockwell, Xudong Lin, Ari Simenauer, Suman R. Das & David E. Wentworth
  4. Comparative Medicine Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA
    Mahnaz Minai, Ian Moore & Marlene Orandle

Authors

  1. Seema S. Lakdawala
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  2. Akila Jayaraman
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  3. Rebecca A. Halpin
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  4. Elaine W. Lamirande
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  5. Angela R. Shih
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  6. Timothy B. Stockwell
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  7. Xudong Lin
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  8. Ari Simenauer
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  9. Christopher T. Hanson
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  10. Leatrice Vogel
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  11. Myeisha Paskel
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  12. Mahnaz Minai
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  13. Ian Moore
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  14. Marlene Orandle
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  15. Suman R. Das
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  16. David E. Wentworth
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  17. Ram Sasisekharan
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  18. Kanta Subbarao
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Contributions

S.S.L., A.J., R.S., D.E.W. and K.S. designed the study. S.S.L., A.J., E.W.L., A.R.S., X. L., A.S., C.T.H, L.V., M.P. and M.M. performed the experiments. S.S.L., R.A.H., T.B.S, I.M., M.O. and S.R.D. analysed the data. S.S.L., K.S., A.J. and R.S. wrote the paper.

Corresponding authors

Correspondence toRam Sasisekharan or Kanta Subbarao.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Amino acids in the receptor binding site of the 2009 H1N1pdm HA that bind to α2,3-linked and α2,6-linked sialic acid glycan receptor.

a–c, Ribbon diagrams of the 2009 H1N1pdm HA receptor binding pocket interacting with an α2,6-linked sialic acid glycan in the pocket (a), an α2,3 H1N1pdm HA with α2,3-linked sialic acid glycan (b), an α2,3 G222D revertant H1N1pdm HA with an α2,6-linked sialic acid glycan (c).

Extended Data Figure 2 Replication of α2,3 H1N1pdm virus in ferret respiratory tract.

a–f, We confirmed that the α2,3 H1N1pdm virus replicated to high titres on days 1, 3, 5 and 7 in different parts of the ferret respiratory tract. Each tissue homogenate is highlighted with a dashed circle; the grey circles represent washes. Each point represents a single animal. The horizontal black line indicates the mean viral titre on a given day.

Extended Data Figure 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 Figure 4 Airborne transmission of α2,3 H1N1pdm virus after 48 h exposure time.

Transmission studies were performed with 4 pairs of animals (8 animals total) in double secure cages with perforated dividers. One ferret in each pair was infected with 106 TCID50 of the indicated virus; a naive ferret (referred to as airborne-contact) was introduced into the adjacent compartment 24 h later. The airborne-contact 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 titres from the nasal secretions are graphed for each infected or airborne-contact animal. The grey shading indicates the exposure time between the infected and airborne-contact animals.

Extended Data Figure 5 Influenza receptor distribution on ferret soft palate.

Haematoxylin and eosin (H&E) staining of the soft palate from an uninfected ferret highlights the nasopharyngeal and oral surfaces. Scale bar, 1.25 mm. a, Areas highlighted in parts b–g are marked with dashed polygons: square, nasopharyngeal surface (b and e); circle, submucosal gland (c and f); and triangle, oral surface (d and g). b–d, H&E staining of these regions, reproduced from Fig. 4a–c, are shown. e–g, Staining with plant lectins specific for α2,6 sialic acid (SNA) and α2,3 sialic acid (MAL II) are shown. Scale bars are 100 μm in images b–g.

Extended Data Figure 6 SC18 staining of ferret respiratory tissues.

a–d, f, g, Sections of ferret trachea (a), lung (b), soft palate (c, d), biopsy of nasal turbinate (NT) 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-linked sialic acids. h, Illustration of ferret head (sectioned along the midline) highlighting the anatomical locations of respiratory epithelium and OLF tissues. Goblet cells on the respiratory epithelium of the soft palate (nasopharyngeal surface) also stained positive for SC18 (d). e, Absence of SC18 staining after sialidase A treatment indicates the high specificity of SC18 for the respiratory epithelium of the soft palate. All scale bars are 100 μm unless indicated.

Extended Data Figure 7 Influenza receptor distribution on pig and human soft palate.

a–c, g–i, 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 acids, respectively. d-f, j-l, Sialidase-A-treated control was run for each sample to ensure specificity of plant lectins and are displayed. 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 submucosal glands of both the pig and human soft palate. Expression of α2,3-linked 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-linked 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, 100 μm.

Extended Data Figure 8 Pathology of the soft palate during infection with α2,3 H1N1pdm virus.

The soft palate was removed from three ferrets infected with α2,3 H1N1pdm virus on 7 dpi. The tissue sections were stained with haematoxylin and eosin. Black arrows indicate the ciliated respiratory epithelium of the soft palate tissue (nasopharyngeal surface). Scale bars, 100 μm in all images.

Extended Data Figure 9 Quasi-species in putative lysine fence.

Deep sequencing analysis of the α2,3 H1N1pdm inoculum revealed a mixed population at four lysine residues surrounding the receptor binding site of the HA protein. The lysine fence was restored in viruses from the nasal wash of airborne-contact animals from 6 days post-exposure (DPE). Each bar represents a single animal, and each amino acid (aa) that contained a quasi-species is indicated.

Extended Data Figure 10 Quasi-species of lysine fence in various ferret respiratory tissue sections.

Deep sequencing of viruses from respiratory tissues of ferrets infected with α2,3 H1N1pdm virus. a–f, Virus populations from the soft palate (a), nasal wash (b), nasal turbinates (c), trachea (d), bronchoalveolar lavage (BAL) (e), or lung sections (f) were analysed 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.

Supplementary information

Supplementary Table 1

This table shows the serology of ferret respiratory droplet transmission. (PDF 92 kb)

Supplementary Table 2

This table contains a list of all amino acid polymorphisms found in HA gene segment through deep sequencing. (XLSX 122 kb)

Supplementary Table 3

This table contains a list of all amino acid polymorphisms found in NA gene segment through deep sequencing. (XLSX 51 kb)

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Lakdawala, S., Jayaraman, A., Halpin, R. et al. The soft palate is an important site of adaptation for transmissible influenza viruses.Nature 526, 122–125 (2015). https://doi.org/10.1038/nature15379

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