Experimental adaptation of an influenza H5 haemagglutinin (HA) confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets methods
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Experimental infection of ferrets
We used 6- to 10-month-old female ferrets (Triple F Farms) that were serologically negative by HI assay for currently circulating human influenza viruses. Six ferrets per group were anesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg per kg of body weight, respectively) and inoculated intranasally with 10 6 PFU (500 µl) of viruses. On days 3 and 6 post-infection, 3 ferrets per group were euthanized for virologic and pathologic examinations. The virus titres in various organs were determined by plaque assays in MDCK cells.
Serologic tests
Serum samples were collected between days 14 and 20 post-infection, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by use of an HI assay with 0.5% turkey red blood cells (TRBCs). Viruses bearing homologous HA were used as antigens for the HI test.
Solid-phase binding assay
Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays with 0.5 % (vol/vol) TRBC. The direct receptor-binding capacity of viruses was examined by use of a solid-phase binding assay as previously described. Microtitre plates (Nunc) were incubated with the sodium salts of sialylglycopolymers [poly-L-glutamic acid backbones containing N -acetylneuraminic acid linked to galactose through either an α2,3 (Neu5Acα2,3Galβ1,4GlcNAcβ1-pAP) or an α2,6 (Neu5Acα2,6Galβ1,4GlcNAcβ1-pAP) bond] in PBS at 4°C overnight. After the glycopolymer solution was removed, the plates were blocked with 0.15 ml...
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We used 6- to 10-month-old female ferrets (Triple F Farms) that were serologically negative by HI assay for currently circulating human influenza viruses. Six ferrets per group were anesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg per kg of body weight, respectively) and inoculated intranasally with 10 6 PFU (500 µl) of viruses. On days 3 and 6 post-infection, 3 ferrets per group were euthanized for virologic and pathologic examinations. The virus titres in various organs were determined by plaque assays in MDCK cells.
Serum samples were collected between days 14 and 20 post-infection, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by use of an HI assay with 0.5% turkey red blood cells (TRBCs). Viruses bearing homologous HA were used as antigens for the HI test.
Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in phosphate-buffered saline (PBS), and ultracentrifuged at 25,000 rpm for 2 h at 4°C. Virus stocks were aliquoted and stored at -80°C. Virus concentrations were determined by using HA assays with 0.5 % (vol/vol) TRBC. The direct receptor-binding capacity of viruses was examined by use of a solid-phase binding assay as previously described. Microtitre plates (Nunc) were incubated with the sodium salts of sialylglycopolymers [poly-L-glutamic acid backbones containing N -acetylneuraminic acid linked to galactose through either an α2,3 (Neu5Acα2,3Galβ1,4GlcNAcβ1-pAP) or an α2,6 (Neu5Acα2,6Galβ1,4GlcNAcβ1-pAP) bond] in PBS at 4°C overnight. After the glycopolymer solution was removed, the plates were blocked with 0.15 ml of PBS containing 4% bovine serum albumin (BSA) at room temperature for 1 h. Following four successive washes with ice-cold PBS, the plates were incubated in a solution containing influenza virus (8 to 32 HA units in PBS) at 4°C overnight. After washing as described above, the plates were incub...
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