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In MDCK cells and primary human airway epithelial cells, CA04 grew to titres comparable to those typically obtained for contemporary human H1N1 influenza viruses (Supplementary Figure A5-4). Confocal, transmission electron and scanning electron microscopy revealed virions of remarkably filamentous shape (Supplementary Figure A5-5), in marked contrast to the spherical shape observed with negatively stained virions ( The biological significance of the morphology of CA04 remains unknown.

To evaluate the pathogenicity of S-OIV in mammalian models, we conducted studies in mice, ferrets, non-human primates and pigs. BALB/c mice intranasally infected with a high dose (>104 plaque forming units (p.f.u.)) of CA04 (Supplementary Figure A5-6) experienced weight loss and those infected with the highest dose of this virus were humanely killed, in contrast to animals infected with a recent human H1N1 virus (A/Kawasaki/UTK-4/09, KUTK-4). The 50% mouse lethal dose (MLD50) was 105.8 p.f.u. for CA04 and .106.6 p.f.u. for KUTK-4. For the additional S-OIV isolates tested, the MLD50 values were >106.4 p.f.u. for Osaka164, >106.6 p.f.u. for WSLH049, 104.5 p.f.u. for WSLH34939 and >105.8 p.f.u. for Net603.

On day 3 after infection of mice, similar titres were detected in nasal turbinates of mice infected with 105 p.f.u. of S-OIVs or KUTK-4 (Supplementary Table A5-2); however, S-OIVs replicated more efficiently in the lungs of infected animals, which may account for the prominent bronchitis and alveolitis with viral antigen on day 3 after infection with CA04 (Supplementary Figure A5-7a, b). On day 6 after infection, virus titres followed a similar trend and the lungs of CA04-infected mice showed bronchitis and alveolitis with viral antigen, although signs of regeneration were apparent (Supplementary Figure A5-7c). We detected viral-antigen-positive bronchial epithelial cells, but not alveolar cells, on day 3 after infection of mice infected with KUTK-4 (Supplementary Figure A5-7e). By day 6, infection in KUTK-4-inoculated mice had progressed to bronchitis and peribronchitis; however, viral antigen was rarely detected in these lesions (Supplementary Figure A5-7f).

There were marked differences in the induction of pro-inflammatory cytokines in the lungs of mice infected with CA04 compared with KUTK-4 (Supplementary Figure A5-8ac). Infection with KUTK-4 resultedin limited induction of pro-inflammatory cytokines/chemokines in the lungs, inmarked contrast to infection withCA04. Increased production of interleukin-10 (IL-10; Supplementary Figure A5-7a) in lungs of CA04-infected mice at day 6 after infection probably reflects a host response to dampen over-exuberant pulmonary inflammation and promote tissue repair. Infection with CA04 led to strong induction of both interferon-γ (IFN-γ) and IL-4 in the lungs. The selective induction of the TH2 cytokine IL-5 in CA04-infected, but not in KUTK-4-infected, mice on day 6 after infection is noteworthy (Supplementary Figure A5-7b), but further studies are needed to understand the relevance of this finding to viral control. IL-17 has been reported to have a role in protection against lethal influenza and also in eliciting

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