Respiratory droplet transmission of H5 avian-human reassortant viruses in ferrets. Groups of three or six ferrets were inoculated intranasally with 10⁶ PFU of pdm09 (a), H5HA/pdm09 (b), or H5HA-mutant/pdm09 (c). One day post-infection, three or six naive ferrets were placed in each adjacent cage. Nasal washes were collected every other day from both inoculated (left panel) and contact (right panel) animals for virus titration. Virus titres in organs were determined by plaque assay in MDCK cells. The lower limit of detection is indicated by the horizontal dashed line.

Virus replication in respiratory organs. Ferrets were infected intranasally with 10⁶ PFU of virus. Three ferrets per group were euthanized on days 3 and 6 after infection for virus titration. Virus titres in nasal turbinates, trachea, and lung were determined by plaque assay in MDCK cells. Horizontal bars show the mean. Asterisks indicate virus titres significantly different from that of pdm09 (Dunnett’s test; P < 0.05).

Pathological analyses of H5 avian-human reassortant viruses. a, Representative histological changes in nasal turbinates from influenza virus-infected ferrets. Three ferrets per group were infected intranasally with 10⁶ PFU of virus, and tissues were collected on day 3 after infection for pathological examination. Uninfected ferret tissues served as negative controls (normal). Left panel, haematoxylin-eosin staining. Right panel, immunohistochemical staining for viral antigen detection (brown staining). Bars, 50 μm. b, Representative histological changes in lungs from influenza virus-infected ferrets. Three ferrets per group were infected intranasally with 10⁶ PFU of virus, and tissues were collected on days 3 and 6 after infection for pathological examination. Left panel, the histological findings in the lungs are represented as schematic diagrams for one of the three animals per group. Cutting aspects of each lung lobe are illustrated with lesion distribution: blue, normal appearance; green, bronchopneumonia; red, alveolitis; and pink, interstitial thickening. Red circles indicate the areas shown in the histology sections. RU, right upper lobe; LU, left upper lobe; RM, right middle lobe; LL, left lower lobe; RL, right lower lobe; Ac, accessory lobe. Middle panel, haematoxylin-eosin staining. Right panel, immunohistochemical staining for viral antigen detection (brown staining). Bronchi are indicated by asterisks “*”. Inserts show a higher magnification of the antigen-positive cells. The lesions were diagnosed as bronchopneumonia, when the lung lesion was observed mainly around the inflammatory or viral antigen-positive bronchi/bronchioles. By contrast, the lesions were diagnosed as alveolitis when bronchi and bronchioles remained intact and inflammation was observed mainly in alveoli. Interstitial thickening was occasionally observed as stimulation by inoculum or secondary to the severe inflammation of adjacent tissue. Bars, 200 μm. c, Pathological severity scores in infected ferrets. To represent comprehensive histological changes, respiratory tissue slides were evaluated by scoring the pathological changes and viral antigen expression levels. The pathological scores were determined for each animal in each group (n = 3/group on days 3 and 6 pi) using the following scoring system: 0 = no pathological change/antigen negative; 1 = affected area (<30%) or only interstitial lesion/rare viral antigens; 2 = affected area (<80%, ≥30%)/moderate viral antigens; 3 = severe lesion ( ≥80%)/many viral antigens; Nasal: pathologic changes in the nasal mucosa, Nasal Ag: viral antigens in the nasal mucosa, Lung: pathologic changes in the lungs, Lung Ag: viral antigens in the lungs. Asterisks indicate virus pathological scores significantly different from that of pdm09 (Dunnett’s test; P < 0.05).

Characterization of the receptor-binding properties of an H5 HA mutant virus possessing four mutations in its HA protein. a, Binding of an H5 HA-mutant virus to sialylglycopolymers in solid-phase binding assays. A human seasonal H1N1 virus, a wild-type avian H5 HA virus, and a mutant H5 HA virus were compared for their ability to bind to sialylglycopolymers containing either α2,3-linked (blue) or α2,6-linked (red) sialic acids. b, Binding of an mutant H5 HA virus to human respiratory tissues. Human, wild-type H5, and mutant H5 HA viruses were incubated with human tissue sections and then stained with the appropriate antibody (anti-human virus antiserum to detect human influenza virus binding or anti-H5 HA antibodies to detect binding by wild-type and mutant H5 HA viruses). All sections were subsequently incubated with fluorescent-labeled secondary antibodies and Hoechst dye (blue). Green stain indicates virus binding.