Supplementary information Methods Cells and viruses. Human isolates (A/Kawasaki/173/01 [H1N1], A/Yokohama/2057/03 [H3N2], and A/Hong Kong/213/03 [H5N1]) were grown in Madin-Darby canine kidney (MDCK) cells maintained in minimal essential medium (MEM) with 5% newborn calf serum, while A/duck/Vietnam/5001/05 (H5N1), A/duck/Mongolia/301/01 (H3N2), and A/duck/Czechoslovakia/56(H4N6) were grown in embryonated chicken eggs. Detection of SAα2,3Gal and SAα2,6Gal in human respiratory tissues. Paraffin-embedded, surgically removed normal human upper-to-lower respiratory tract tissues (bronchial and lung, eight patients; tracheal, nasal, and pharyngeal; three patients) were cut into 5-µm thick sections with a microtome, mounted on 3-aminopropyltrethoxy-silane (APS)-coated slides (Matsunami Glass Ind., Ltd., Tokyo), deparaffinized in xylene and rehydrated by alcohol. For detection of sialyloligosaccharides reactive with SAα2,3Gal- or SAα2,6Gal-specific lectins, the sections were incubated with 250 µl of FITC-labeled Sambucus nigra (SNA) lectin (Vector Laboratories, Burlingame, CA) and biotinylated Maackia amurensis (MAA) lectin (Vector Laboratories) overnight at 4 o C. After three washes with Tris-buffered saline (TBS, ph 7.6), the sections were incubated with Alexa Fluor 594-conjugated streptavidin, (Molecular Probes, Inc., Eugene, OR) for 2 hours at room temperature. The sections were counterstained with 4,6-diamino-2- phenylindole, dihydrochloride (DAPI; Dojindo Molecular Technologies, Inc., Kumamoto). After three washes with TBS, the sections were mounted on cover glass and observed with a fluorescence microscope (ECLIPSE TE300 with a fluorescence equipment mercury set, Nikon Co., Tokyo). Photos were taken with a digital microscope camera (Olympus DP70, Olympus
Optical Co., Ltd., Tokyo). Human research ethics approval for all human specimens was obtained from the Tohoku University Office of Research Ethics. Double-immunohistochemical/lectin staining of human lung sections. To determine the types of cells reactive with MAA lectin, we double-stained sections for surfactant protein-a (SP-A), a marker for type II alveolar cells, in conjunction with MAA lectin. Briefly, deparaffinized sections were incubated with anti-human SP-A mouse antibody (DakoCytomation Co. Ltd., Kyoto) and biotinylated MAA lectin overnight at 4 o C. The sections were next incubated with FITCconjugated antimouse IgG (Molecular Probes) and Alexa Fluor 594-conjugated streptavidin for 2 hours at room temperature. They were then counterstained with DAPI. Binding of influenza A viruses to bronchial and alveolar cells. Virus was labelled with fluorescent protein labelling kits (Pierce, Rockford, Illinois). Fresh surgical human lung specimens were embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical Co., Ltd., Tokyo) and frozen on a dry ice block. Sections of each tissue (8 µm) were cut with a cryostat (CM 3050S, Leica microsystems, Nussloch, Germany), and air dried. After washing with MEM, the sections were incubated with fluorescent-labeled virus (equivalent to 10 9 plaque-forming units) overnight at 4 o C, washed, counterstained with DAPI, and processed for fluorescence detection. Infection of human lung tissue with influenza A viruses. Fresh surgically removed normal human lung specimens that contained bronchi and alveoli were cut into 0.5-cm 3 cubes, washed with culture medium (F-12K nutrient mixture with 15% FCS, L-glutamine, and antibiotics), and incubated with virus (~10 8 infectious viruses/ml) at 37 o C in the medium. Twelve hours post-
infection, the tissue blocks were fixed with 10% neutral buffered formalin and processed for routine paraffin embedding and immunohistochemical analysis with mouse anti-influenza A virus nucleoprotein antibody. The reactions were visualized by using a two-step dextran polymer system (DAKO) and 3,3'-diamino benzidine (DAB). These experiments were performed with tissues from three patients. The results were comparable, so that only findings from one set of specimens are shown. Supplementary figures
Supplementary Figure 1 Reactivity of human respiratory tissues with sialic acid linkagespecific lectins. a, Nasal mucosa; b, paranasal sinuses; c, pharynx; d, trachea; e, bronchus; f, bronchiole; g, alveolus; Res, respiratory bronchiole; Ter, terminal bronchiole; Al, alveolus. Green, reaction with SNA lectin, indicating the presence of SAα2,6Gal. Red, reaction with MAA lectin, indicating the presence of SAα2,3Gal. Cells were counterstained with DAPI. Tissue samples obtained from different individuals gave comparable results. Supplementary Figure 2 Identification of cells reactive with MAA lectin in human lungs as alveolar type II cells. Double immunohistochemistry with SAα2,3Gal-specific MAA lectin (red) and antisurfactant protein A (SP-A) antibody (green). Many of the MAA-positive alveolar cells expressed SP-A secretory vesicles in the cytoplasm.
Supplementary Figure 3 Binding of influenza A viruses to human lung tissue. Binding of FITC-labeled human A/Kawasaki/173/01 (H1N1) virus (green; a, b), rhodamine-labeled A/duck/Mongolia/301/01 (H3N2) (reddish pink; c, d), and rhodamine-labeled A/Hong Kong/213/03 (H5N1; reddish pink; e, f) to bronchiolar epithelial cells (a, c, e) and alveolar cells (b, d, f). The results with other human viruses, A/Yokohama/2057/03 (H3N2) and A/Kawasaki/176/02 (H1N1) were similar to those with A/Kawasaki/173/01, while those with other avian viruses, A/duck/Czechoslovakia/56 (H4N6) and A/duck/Vietnam/5001/05 (H5N1) were similar to those with A/duck/Mongolia/301/01 (not shown).
a. b. c. d. Supplementary Figure 4 Infection of human lung tissue by H5N1 influenza A viruses. a. Bronchial epithelial cells are not infected with A/duck/Vietnam/5001/05 virus preferentially recognizing SAα2,3Gal. b, Infection of alveolar cells by this duck virus. c, Infection of bronchial epithelial cells with A/Hong Kong/213/03 recognizing both SAα2,3Gal and SAα2,6Gal. d, Infection of alveolar cells with this dual-binding H5N1 human virus. Infected cells are stained in brown.