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A5
IN VITRO AND IN VIVO CHARACTERIZATION OF NEW SWINE- ORIGIN H1N1 INFLUENZA VIRUSES23

Yasushi Itoh,24 Kyoko Shinya,25 Maki Kiso,26 Tokiko Watanabe,27 Yoshihiro Sakoda,28 Masato Hatta,27 Yukiko Muramoto,29 Daisuke Tamura,26 Yuko Sakai-Tagawa,26 Takeshi Noda,30 Saori Sakabe,26 Masaki Imai,27 Yasuko Hatta,27 Shinji Watanabe,27 Chengjun Li,27 Shinya Yamada,26 Ken Fujii,26 Shin Murakami,26 Hirotaka Imai,26 Satoshi Kakugawa,26 Mutsumi Ito,26 Ryo Takano,26 Kiyoko Iwatsuki-Horimoto,26 Masayuki Shimojima,26 Taisuke Horimoto,26 Hideo Goto,26 Kei Takahashi,26 Akiko Makino,25 Hirohito Ishigaki,24 Misako Nakayama,24 Masatoshi Okamatsu,28 Kazuo Takahashi,31 David Warshauer,32 Peter A. Shult,32 Reiko Saito,33 Hiroshi Suzuki,33 Yousuke Furuta,34 Makoto Yamashita,35 Keiko Mitamura,36 Kunio Nakano,36 Morio Nakamura,36 Rebecca Brockman-Schneider,37 Hiroshi Mitamura,38 Masahiko Yamazaki,39 Norio Sugaya,40 M. Suresh,27 Makoto Ozawa,27,30 Gabriele Neumann,27 James Gern,37 Hiroshi Kida,28 Kazumasa Ogasawara,24 and Yoshihiro Kawaoka25,26,27,29,30,41

23

Reprinted with permission from Nature 460(7258):1021-1025.

24

Department of Pathology, Shiga University of Medical Science, Ohtsu, Shiga 520-2192, Japan.

25

Department of Microbiology and Infectious Diseases, Kobe University, Hyogo 650-0017, Japan.

26

Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

27

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA.

28

Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.

29

ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan.

30

Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

31

Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan.

32

Wisconsin State Laboratory of Hygiene, Madison, Wisconsin 53706, USA.

33

Department of Public Health, Niigata University, Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan.

34

Toyama Chemical Co., Ltd., Toyama 930-8508, Japan.

35

Daiichi Sankyo Co Ltd, Shinagawa, Tokyo 140–8710, Japan.

36

Eiju General Hospital, Tokyo 110-8654, Japan.

37

School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53792, USA.

38

Department of Internal Medicine, Mitamura Clinic, Shizuoka 413-0103, Japan.

39

Department of Pediatrics, Zama Children’s Clinic, Kanagawa 228-0023, Japan.

40

Keiyu Hospital, Kanagawa 220-0012, Japan.

41

Creative Research Initiative, Sousei, Hokkaido University, Sapporo 060-0818, Japan.



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155 APPENDIX A A5 IN VITRO AND IN VIVO CHARACTERIZATION OF NEW SWINE-ORIGIN H1N1 INFLUENZA VIRUSES23 Yasushi Itoh,24 Kyoko Shinya,25 Maki Kiso,26 Tokiko Watanabe,27 Yoshihiro Sakoda,28 Masato Hatta,27 Yukiko Muramoto,29 Daisuke Tamura,26 Yuko Sakai-Tagawa,26 Takeshi Noda,30 Saori Sakabe,26 Masaki Imai,27 Yasuko Hatta,27 Shinji Watanabe,27 Chengjun Li,27 Shinya Yamada,26 Ken Fujii,26 Shin Murakami,26 Hirotaka Imai,26 Satoshi Kakugawa,26 Mutsumi Ito,26 Ryo Takano,26 Kiyoko Iwatsuki-Horimoto,26 Masayuki Shimojima,26 Taisuke Horimoto,26 Hideo Goto,26 Kei Takahashi,26 Akiko Makino,25 Hirohito Ishigaki,24 Misako Nakayama,24 Masatoshi Okamatsu,28 Kazuo Takahashi,31 David Warshauer,32 Peter A. Shult,32 Reiko Saito,33 Hiroshi Suzuki,33 Yousuke Furuta,34 Makoto Yamashita,35 Keiko Mitamura,36 Kunio Nakano,36 Morio Nakamura,36 Rebecca Brockman-Schneider,37 Hiroshi Mitamura,38 Masahiko Yamazaki,39 Norio Sugaya,40 M. Suresh,27 Makoto Ozawa,27,30 Gabriele Neumann,27 James Gern,37 Hiroshi Kida,28 Kazumasa Ogasawara,24 and Yoshihiro Kawaoka25,26,27,29,30,41 23 Reprinted with permission from Nature 460(7258):1021-1025. 24 Department of Pathology, Shiga University of Medical Science, Ohtsu, Shiga 520-2192, Japan. 25Department of Microbiology and Infectious Diseases, Kobe University, Hyogo 650-0017, Japan. 26 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 27 Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. 28 Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. 29 ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan. 30 Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 31 Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan. 32 Wisconsin State Laboratory of Hygiene, Madison, Wisconsin 53706, USA. 33 Department of Public Health, Niigata University, Graduate School of Medical and Dental Sci- ences, Niigata 951-8510, Japan. 34 Toyama Chemical Co., Ltd., Toyama 930-8508, Japan. 35 Daiichi Sankyo Co Ltd, Shinagawa, Tokyo 140–8710, Japan. 36 Eiju General Hospital, Tokyo 110-8654, Japan. 37 School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53792, USA. 38 Department of Internal Medicine, Mitamura Clinic, Shizuoka 413-0103, Japan. 39 Department of Pediatrics, Zama Children’s Clinic, Kanagawa 228-0023, Japan. 40 Keiyu Hospital, Kanagawa 220-0012, Japan. 41 Creative Research Initiative, Sousei, Hokkaido University, Sapporo 060-0818, Japan.

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156 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global econ- omy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses (Novel Swine-Origin Influenza A [H1N1] Virus Investigation Team, 2009). Most human infections with swine origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic. Sequence analyses of recently emerged swine-origin H1N1 viruses (S-OIVs) revealed the absence of markers associated with high pathogenicity in avian and/or mammalian species, such as a multibasic haemagglutinin (HA) cleavage site (Kawaoka and Webster, 1988) or lysine at position 627 of the PB2 pro- tein (Hatta et al., 2001). To characterize the new viruses in vitro and in vivo, we amplified the following S-OIVs in Madin–Darby canine kidney (MDCK) cells: A/California/04/09 (CA04), A/Wisconsin/WSLH049/09 (WSLH049), A/Wisconsin/WSLH34939/09 (WSLH34939), A/Netherlands/603/09 (Net603) and A/Osaka/164/09 (Osaka164). WSLH34939 was isolated from a patient who required hospitalization, whereas the remaining viruses were isolated from mild cases. These viruses represent the currently recognized neuraminidase (NA) variants among S-OIVs: CA04, NA-106V, NA-248N; Osaka164, NA-106I, NA-248N; WSLH049, NA-106I, NA-248D; WSLH34939, NA-106I, NA-248D; and Net603, NA-106V, NA-248N.

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157 APPENDIX A In MDCK cells and primary human airway epithelial cells, CA04 grew to titres comparable to those typically obtained for contemporary human H1N1 influ- enza 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 (http://www.cdc.gov/h1n1flu/images.htm). 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 (Supple- mentary 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 tur- binates 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 cyto- kines in the lungs of mice infected with CA04 compared with KUTK-4 (Supple- mentary Figure A5-8a–c). 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; Supplemen- tary Figure A5-7a) in lungs of CA04-infected mice at day 6 after infection prob- ably 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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158 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC inflammatory responses (Iwakura et al., 2008; Hamada et al., 2009); however, the enhanced viral replication and lung pathology observed in CA04-infected mice was not linked to dysregulated IL-17 production. Cynomolgus macaques (Macaca fascicularis) have been used to study highly pathogenic avian H5N1 viruses (Baskin et al., 2009; Rimmelzwaan et al., 2001) and the 1918 pandemic virus (Kobasa et al., 2007). Infection of cynomolgus macaques with CA04 (see Methods for detailed procedures) resulted in a more prominent increase in body temperature than infection with KUTK-4 (Supplemen- tary Fig. A5-9). This difference might originate from the observed differences in virus titres (Table A5-1 and Supplementary Table A5-3). No remarkable difference in body weight loss was found between the two groups (data not shown). CA04 replicated efficiently in the lungs and other respiratory organs of infected animals, similar to highly pathogenic influenza viruses (Baskin et al., 2009; Kobasa et al., 2007) (Table A5-1). By contrast, conventional human influenza viruses are typi- cally limited in their replicative ability in the lungs of infected primates (Baskin et al., 2009; Kobasa et al., 2007) (Table A5-1), although a seasonal H1N1 virus was isolated from one animal on day 7 after infection. Pathological examina- tion revealed that CA04 caused more severe lung lesions than did KUTK-4 (Fig. A5-1 and Supplementary Fig. A5-10). On day 3 after infection with CA04, alveolar spaces were occupied by oedematous exudate and inflammatory infil- trates (Fig. A5-1a, b); severe thickening of alveolar walls was also observed (Fig. A5-1b). Viral-antigen-positive cells were distributed in the inflammatory lesions, and many of these cells were elongated with thin cytoplasm and hemming around the alveolar wall, indicating type I pneumocytes (Fig. A5-1c). In addi- tion to type I pneumocytes, CA04 viral antigens were also detected in consider- able numbers of cuboidal, cytokeratin-positive cells, hence identified as type II pneumocytes (Fig. A5-1d and Supplementary Fig. A5-11), as has been reported for highly pathogenic avian H5N1 influenza viruses6. Upon infection with KUTK-4, large sections of infected lungs showed thickening of the alveolar wall on day 3 after infection (Fig. A5-1e). Although the infiltration of inflammatory cells was prominent at the alveolar wall (Fig. A5-1f), viral antigens were sparse and detected in type I (but not type II) pneumocytes (Fig. A5-1g). By contrast, the lungs of non-infected animals show clear alveolar spaces (Fig. A5-1h). On day 7 after infection, lung pathology remained more severe for CA04- than for KUTK-4-infected lungs (Supplementary Fig. A5-10), although regen- erative changes were seen for CA04. Nonetheless, considerable numbers of antigen-positive cells were still detectable (Supplementary Fig. A5-10c). Collec- tively, these findings demonstrate that CA04 causes more severe lung lesions in non-human primates than does a contemporary human influenza virus. Induction of pro-inflammatory cytokines/chemokines in the lungs of CA04-infected macaques was variable at day 3 after infection (Supplementary Fig. A5-12). However, consistent with persisting lung pathology and inflamma- tion on day 7 after infection, the levels of MCP-1, MIP-1α, IL-6 and IL-18 were markedly higher in the lungs of two of three CA04-infected macaques.

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TABLE A5-1 Virus Titres in Organs of Infected Cynomolgus Macaques Organ A/California/04/09 (H1N1) A/Kawasaki/UTK-4/09 (H1N1) Day 3 after infection Day 7 after infection Day 3 after infection Day 7 after infection 1 2 3 4 5 6 7 8 9 10 11 12 Nasal mucosa 4.7 3.3 — — — — — — — — — — Oro/nasopharynx 6.3 4.4 4.7 — 7.9 — — — 4.3 — — 4.8 Tonsil 6.4 — — — 7.1 — — — 2.8 — — 3.0 Trachea 5.9 2.0 5.6 — — — 2.0 4.1 — 3.7 — 5.4 Bronchus (right) 5.7 2.9 4.3 — 5.1 — — 2.5 — 3.5 — 3.8 Bronchus (left) 5.9 — 6.1 — 5.1 — — — — 3.3 — 5.1 Lung (upper right) 5.7 5.6 4.5 — — — 2.7 — — — — — Lung (middle right) 5.6 6.4 6.9 — — — 2.3 2.6 2.5 — — — Lung (lower right) 6.1 4.5 6.0 — — — 2.6 2.6 — — — 3.4 Lung (Upper left) 4.7 4.3 6.4 — — — — — — — — — Lung (middle left) 5.8 4.3 6.3 — — — — — — — — — Lung (lower left) 6.7 4.5 6.6 — — — — — — — — 2.3 Conjunctiva 3.6 — — — — — — — — — — — Cynomolgus macaques were inoculated with 107.4 p.f.u. of virus (6.7 ml) through multiple routes (see Methods). Three macaques per group were killed on days 3 and 7 after infection for virus titration. No virus was recovered from lymph nodes (chest), heart, spleen, kidneys or liver of any of the animals. A dash indicates that virus was not detected (detection limit: 2 log10 p.f.u.g–1). Numbers 1-12 indicate animal identification number. Values indicate virus titre (mean log10 p.f.u.g–1). 159

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160 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC CA04 KUTK-4 Left Right Left Right Normal lung FIGURE A5-1 Pathological examination of the lungs of infected cynomolgus macaques. A-h Representative pathological images of CA04-infected (macaque no. 1, a-d), KUTK-4 Figure WO-8 and A5-1 infected (macaque no. 7, e-g) and mock-infected (h) lungs on day 3 after infection. One or R01627 two sections per lung lobe were examined. Representative findings are shown to depict the distribution of lesions inuneditable bitmapped image the sections (shown as cross-sections placed next to illustrations of ach lung lobe), with or without viral text replaced brown, severe lung lesion con- with antigen, as follows: taining moderate to many viral-antigen-positive cells; pink, mild lung lesions containing a few viral-antigen-positive cells; blue, lung lesions with alveolar wall thickening, with remaining air spaces unaffected. Original magnification: a, e, h, ×40; b-d-f, g, ×400. Ferrets are widely accepted as a suitable small-animal model for influenza virus pathogenicity and transmissibility studies. Infection of ferrets with S-OIVs or KUTK-4 did not cause marked changes in body temperature or weight in any group (data not shown). Although all test viruses were detected in nasal turbinates at similar titres on day 3 after infection (Supplementary Table A5-4), S-OIVs replicated to higher titres in trachea and lungs. Pathological examination detected similar levels of viral antigen in the nasal mucosa of both CA04- and KUTK-4-infected ferrets (Supplementary Fig. A5-13a and e). However, the lungs of CA04-infected ferrets showed more severe broncho-

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161 APPENDIX A pneumonia with prominent viral antigen expression in the peribronchial glands and a few alveolar cells (Supplementary Fig. A5-13b–d) on day 3 after infection. By contrast, most of the lung appeared normal after infection with KUTK-4 (Supplementary Fig. A5-13f and g). Thus, in all three mammalian models tested, CA04 seemed to be more pathogenic than a contemporary human H1N1 virus, KUTK-4. Efficient human-to-human transmission is a critical feature of pandemic influenza viruses. To assess the transmissibility of CA04, naive ferrets in perfo- rated cages were placed next to ferrets inoculated with 106 p.f.u. of CA04 (see Methods for detailed procedures). This experimental setting allows for aerosol transmission (that is, the exchange of respiratory droplets between the inoculated and noninoculated ferrets) but prevents transmission by direct and indirect con- tact. All three contact ferrets were positive for CA04 virus on days 3 and 5 after infection (Supplementary Table A5-5). This transmission pattern is comparable to those of two human control influenza viruses that are known to transmit among ferrets: KUTK-4 and A/Victoria/3/75 (H3N2) (Maines et al., 2006). By contrast, an avian influenza virus (A/duck/Alberta/35/76; H1N1) did not transmit (Supple- mentary Table A5-5). Genetic analysis suggests that S-OIV originated in pigs (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009). However, there were no confirmed influenza virus outbreaks in Central American pigs before the reported S-OIV infections in humans. To assess S-OIV replication in pigs, we inoculated specific-pathogen-free miniature pigs, which are easier to manage, with CA04 or a classical swine influenza virus (A/swine/Hokkaido/2/81, H1N1). No signs of dis- ease were observed (data not shown), although both viruses replicated efficiently in the respiratory organs of these animals (Supplementary Tables A5-6 and A5-7). Slightly higher titres of CA04 were detected in lungs on day 3 after infection, which is supported by pathological findings that show more apparent bronchitis and bronchiolitis in pigs infected with CA04 (Supplementary Fig. A5-14). The asymp- tomatic infection of CA04, despite efficient virus replication, might explain the lack of reports of S-OIV outbreaks in pigs before virus transmission to humans. Antiviral compounds are the first line of defence against pandemic influenza viruses. Sequence analysis suggests that S-OIVs are resistant to ion channel inhibi- tors such as amantadine and rimantadine (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009). We therefore tested the licensed neuraminidase inhibitors oseltamivir and zanamivir, the experimental neuraminidase inhibitor R-125489 (the active form of CS-8958 [Yamashita et al., 2009]) and the experi- mental compound T-705 (a broad-spectrum viral RNA polymerase inhibitor [Furuta et al., 2002]) for their efficacy against CA04. In cell culture, CA04 was highly susceptible to all compounds tested (Supplementary Table A5-8), as were the human H1N1 control viruses A/Kawasaki/UTK-23/08 and KUTK-4, with the exception of the known oseltamivir resistance of KUTK-4. Comparable sensitivi- ties were also found in an enzymatic neuraminidase inhibition assay (Hayden et

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162 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC 8 Day 3 7 Virus titre (log10 p.f.u. g –1) 6 5 4 3 2 A/Kawasaki/UTK-4/09 A/Kawasaki/UTK-23/08 A/California/04/09 (H1N1) (H1N1) (H1N1) Zanamivir (8 mg kg –1) Control Oseltamivir (8 mg kg –1) CS-8958 (0.7 mg kg –1) Oseltamivir (80 mg kg –1) T-705 (60 mg kg –1) Zanamivir (0.8 mg kg ) T-705 (300 mg kg –1) –1 8 Day 6 7 Virus titre (log10 p.f.u. g –1) 6 5 4 3 2 A/California/04/09 A/Kawasaki/UTK-4/09 A/Kawasaki/UTK-23/08 (H1N1) (H1N1) (H1N1) FIGURE A5-2 CA04 sensitivity to antiviral compounds in mice. Mice were intranasally inoculated with 104 p.f.u. (50 μl) of CA04, KUTK-4 or A/Kawasaki/UTK-23/08 (H1N1). At 1 h after infection, mice were administered oseltamivir phosphate, zanamivir, CS-8958, T-705, or distilled water and PBS (control). Three mice per group were killed on days 3 and 6 after infection and the virus titres in lungs were determined by plaque assays in MDCK cells; results are reported as means ± s.d. The statistical significance of differences in lung virus titres of control mice and those treated with antivirals were assessed by use of the Student’s t-test (asterisk, P < 0.05; double asterisk, P < 0.01). Figure WO-10 and A5-2 R01627 bitmapped image with type replaced

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163 APPENDIX A al., 2000) (Supplementary Table A5-9) and in mice (Fig. A5-2), consistent with observations in clinical settings. A recent report suggested that 33% of individuals over 60 years of age had neutralizing antibodies to CA04 (http://www.cdc.gov/mmwr/preview/mmwrhtml/ mm5819a1.htm; Morbidity and Mortality Weekly Report, Centers for Disease Control and Prevention), probably due to previous exposure to antigenically similar H1N1 viruses. In fact, both the human H1N1 viruses that circulated until 1957 and the classical swine virus HA gene of S-OIVs are descendants of the 1918 pandemic virus, possibly explaining their antigenic relatedness. In 1977, H1N1 viruses re-emerged that were genetically and antigenically very closely related to viruses circulating in the 1950s (Nakajima et al., 1978) and should thus have elicited neutralizing antibodies to CA04 among younger age groups; however, this does not seem to be the case, according to the above described report. To resolve this puzzling finding, we assessed the neutralizing activities of sera collected from a broad range of age groups against CA04 and KUTK-4. We used two sets of donor sera, collected in 1999 from residents and workers in a nursing home (donor set 1), and in April 2009 from workers and patients in a hospital (donor set 2). High neutralizing activity against KUTK-4 was detected for many sera in donor set 2 (Fig. A5-3), but not for sera in donor set 1, probably because these sera were collected before the emergence of the current human H1N1 viruses. Interestingly, with few exceptions, no appreciable neutralizing antibodies against CA04 were found for individuals born after 1920; however, many of those born before 1918 had high neutralizing antibody titres (individual neutralizing antibody titres are shown in Supplementary Table A5-9). These data indicate that infection with the 1918 pandemic virus or closely related human H1N1 viruses, but not infection with antigenically divergent human H1N1 viruses circulating in the 1920s to 1950s, and again since 1977, elicited neutralizing antibodies to S-OIVs. Our findings indicate that S-OIVs are more pathogenic in mammalian models than seasonal H1N1 influenza viruses. In fact, the ability of CA04 to replicate in the lungs of mice, ferrets and non-human primates, and to cause appreciable pathology in this organ, is reminiscent of infections with highly pathogenic H5N1 influenza viruses (Peiris et al., 2004), as acknowledged in a recent report by the World Health Organization (http://www.who.int/wer/2009/ wer8421/en/index.html). We therefore speculate that the high replicative ability of S-OIVs might contribute to a viral pneumonia characterized by diffuse alveolar damage that contributes to hospitalizations and fatal cases where no other under - lying health issues exist (http://www.who.int/wer/2009/wer8421/en/index.html). In addition, sustained person-to-person transmission might result in the emer- gence of more pathogenic variants, as observed with the 1918 pandemic virus (reviewed in Wright et al., 2007). Furthermore, S-OIVs may acquire resistance to oseltamivir through mutations in their NA gene (as recently witnessed with human H1N1 viruses [Moscona, 2009]), or through reassortment with co-circulating,

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164 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC FIGURE A5-3 Neutralization activities in human sera against viruses. Human sera of donor groups 1 (collected in 1999) and 2 (collected in April and May of 2009) were sub- jected to neutralization assays with CA04 and KUTK-4. Because the sera of donor group 1 were collected in 1999, little neutralization activity was expected against KUTK-4, which was isolated in 2009. Figure WO-9 and A5-3 R01627 oseltamivir-resistant seasonal human bitmapped image uneditable H1N1 viruses. Collectively, our findings are a reminder that S-OIVs have not yet garnered a place in history, but may still do so, as the pandemic caused by these viruses has the potential to produce a significant impact on human health and the global economy. Methods Summary Viruses and Cells All swine-origin H1N1 viruses were isolated and passaged in MDCK cells to produce viral stocks. The viruses and their passage histories are described in Methods. All experiments with S-OIVs were performed in approved enhanced biosafety level 3 (BSL3) containment laboratories.

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165 APPENDIX A MDCK cells and MDCK cells overexpressing the β-galactoside α2,6- sialyltransferase I gene (Hatakeyama et al., 2005) were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum. Human airway epithelial (HAE) cells were obtained from residual surgical tissue trimmed from lungs during the process of transplantation. The bronchial specimens were dissected and enzymatically digested, and monolayers of HAE cells were iso- lated, cultured and differentiated as previously described (Jakiela et al., 2008). Animals Five- and six-week-old female BALB/c mice (Jackson Laboratory and Japan SLC Inc.), approximately three-to-four-year-old cynomolgus macaques (Ina Research Inc.), five-to-eight-month-old male ferrets (Marshall Farms and Triple F Farms) and two-month-old female specific-pathogen-free miniature pigs (Nippon Institute for Biological Science) were used according to approved protocols for the care and use of animals. Detailed procedures are provided in Methods. Antiviral Sensitivity of Viruses in Mice Five-week-old female BALB/c mice (Japan SLC Inc.; groups of six) were anaesthetized with sevoflurane and intranasally inoculated with 104 p.f.u. (vol- ume, 50 μl) of CA04, KUTK-4, or A/Kawasaki/UTK-23/08 (H1N1). At 1 h after infection, mice were administered antiviral compounds as described in detail in Methods. Three mice per group were killed on days 3 or 6 after infection and the virus titres in lungs were determined by plaque assays in MDCK cells. Methods Viruses A/California/04/09 (H1N1; CA04) was provided by the Centers for Disease Control (CDC). A/Wisconsin/WSLH049/09 (H1N1) was isolated from a patient with mild symptoms, whereas A/Wisconsin/WSLH34939/09 (H1N1) was isolated from a hospitalized patient. A/Netherlands/603/09 (H1N1) was isolated from a patient with mild symptoms and was provided by R. Fouchier. A/Osaka/164/09 (H1N1) was also isolated from a patient with mild symptoms. The following influenza viruses served as controls: A/Kawasaki/UTK-4/09 (H1N1; KUTK-4; passaged twice in MDCK cells), an oseltamivir-resistant sea- sonal human virus; A/WSN/33 (H1N1; generated by reverse genetics and passaged twice in MDCK cells), a typical spherical influenza virus (Noda et al., 2006); A/Kawasaki/UTK-23/08 (H1N1; passaged twice in MDCK cells), an oseltamivir- sensitive seasonal human virus; A/Victoria/3/75 (H3N2; passaged several times in eggs after it was obtained from the CDC), a human virus; A/swine/Hokkaido/2/81

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180 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC FIGURE A5-9 Body temperatures of infected cynomolgus macaques. Six macaques per group were inoculated with 107.4 PFU (total volume: 6.7 ml) of CA04 (red #1-6) or KUTK-4 (blue, #7-12) through multiple routes (see Supplementary materials and meth- ods). Temperatures were monitored every 15 minutes by telemetry probes implanted in Figure A5-9 the peritoneal cavities. The periodic sharp reduction in body temperatures on days 0, 1, R01627 3, and 7 was caused by anesthesia required for sampling. Monkeys #1-3 and #7-9 were uneditable bitmapped image euthanized on day 3.

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181 APPENDIX A FIGURE A5-10 Pathological findings in infected cynomolgus macaques. Shown are representative pathological findings in the lungs of cynomolgus macaques on day 7 post infection with CA04 (macaque #5, a-c) or KUTK-4 (macaque #11, d-f) (upper portion). Schematic figures summarize the distribution of lesions, with or without viral antigen, in the lungs of the remaining virus-inoculated macaques. Colors: green, severe lung lesions where alveolar spaces were filled with edema fluid, inflammatory cells, or cell debris; brown, severe lung lesions containingFigure A5-10 viral antigen-positive cells; pink, moderate to many mild lung lesions containing a few viral R01627 antigen-positive cells; blue, lung lesions where uneditable bitmapped image severe alveolar wall thickening was prominent, but air spaces were preserved. (a) Alveolar spaces were not clear because of inflammatory exudate. (b) Large areas of affected lung contained accumulated cell debris, inflammatory infiltrates, fibrin, and edema fluid; alveo- lar walls were thickened by infiltration of inflammatory cells. ( c) Viral antigen-positive cells were detected extensively in lung lesions in some areas. (d) In most areas, alveolar spaces were still clear, although thickening of the alveolar walls was apparent. ( e) Most lung lesions consisted of thickening of alveolar walls by mononuclear cells. ( f) A few antigen-positive cells were detected in lung lesions.

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182 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC FIGURE A5-11 Detection of viral antigens in type II pneumocytes in the lungs of CA04-infected cynomolgus macaques. On day 3 post-infection, cells were stained with anti-cytokeratin (N1590, DAKO) antibody (a; red) and anti-influenza (H1N1) antibody (b; green). The nucleus was stained with DAPI (c). Considerable amounts of viral antigen were detected in type II pneumocytes (d). Figure A5-11 R01627 uneditable bitmapped image

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183 APPENDIX A FIGURE A5-12 Pro-inflammatory cytokine/chemokine responses in the lungs of infected cynomolgus macaques. The concentrations of various cytokines/chemokines in the lungs of infected cynomolgus macaques on day 3 post-infection were measured by protein array analysis with the MILLIPLEX MAP Non-human Primate Cytokine/Chemokine Panel – Premixed 23-Plex (Millipore, Bedford, MA). We did not detect IL-4, IL-17, or TNFα in the lungs or G-CSF and IL-4 in the sera.

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184 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC FIGURE A5-13 Pathological findings in infected ferrets. CA04-omfected ferret lungs showed severe and wide lung lesions with viral antigen on day 3 pi and without viral antigen on day 6 pi. KUTK-4-infected ferret lungs showed limited lung lesopns with viral antigen on days 3 and 6 pi. Representative pathological findings of nasal mucosa and lungs of CA04-(a-d), and KUTK-4-(e-g) infected ferrets on days 3 and 6 pi. (a) Extensive viral antigen present at the nasal epithelium on day 3 pi in CA04-infected Figure A5-13 ferret. (b) and (c) In the lungs, viral antigen was mainly detected in the peribroncial R01627 glands with severe peribronchitis and bronchopneumonia (d) Sparse viral antigen was detected within alveolar lesions. (e) bitmapped image expression at the nasal uneditable Extensive viral antigen mucosa on day 3 pi in KUTK-4-infected ferret. ( f) Most of the lung was not affected by viral infection in KUTK-4-infected ferrets. (g) Most of the peribronchial gland appeared normal in KUTK-4-infected ferret lungs at day 6 pi. Colors: green, severe lung lesions where alveolar spaces were filled with edema fluid, inflammatory cells, or cell debris; brown, severe lung lesions containing moderate to many viral antigen-positive cells.

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185 APPENDIX A FIGURE A5-14 Pathological findings in infected miniature pigs. Shown are representa- tive pathological findings for the lungs of miniature pigs on day 3 post infection with CA04 (miniature pig #1, a-c) or A/swine/Hokkaido/2/81 (H1N1) (miniature pig #5, d-f). Figure A5-14 The distribution of viral antigen (brown) is shown in the schematic figures. (a) The lumens R01627 of the bronchus and bronchioles were filled with inflammatory infiltrates. (b) Viral antigen was detected along bronchus and bronchiole. (c) Viral antigen was mainly detected in uneditable bitmapped image remained clear with epithelial cells and desquamated cells. (d) The bronchial lumen limited inflammatory reactions. (e) and (f) Few antigen-positive cells were detected at the epithelium, with minimum inflammatory reaction. The areas delineated by green boxes in the schematic diagrams correspond to the histopathological sections.

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186 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC TABLE A5-2 Virus Titres in Organs of Infected Micea Virus titres (mean log10 PFU ± SD/g) in: Virus Nasal turbinates Lungs 6.6+0.2 7.8+0.03 A/California/04/09 (H1N1) Day 3 5.4+0.6 6.8+0.01 Day 6 6.6+0.2 6.8+0.3 A/Netherlands/603/09 (H1N1) Day 3 5.5+0.2 6.2+0.2 Day 6 6.7+0.2 7.2+0.2 A/Wisconsin/WSLH049/09 (H1N1) Day 3 6.3+0.2 6.5+0.1 Day 6 7.1+0.2 7.7+0.2 A/Wisconsin/WSLH34939/09 (H1N1) Day 3 5.9+0.3 6.9+0.5 Day 6 6.3+0.7 7.2+0.1 A/Osaka/164/09 (H1N1) Day 3 3.8+1.3 6.5+0.4 Day 6 6.3+0.2 6.4+0.3 A/Kawasaki/UTK-4/09 (H1N1) Day 3 5.0+0.3 4.6+0.4 Day 6 aBALB/c mice were intranasally infected with 105 PFU (50 μl) of virus. Three mice from each group were euthanized on days 3 and 6 pi for virus titration. None of the viruses tested was recovered from the spleens, kidneys, brains, colons, or livers of infected animals. TABLE A5-3 Virus Titres in Respiratory Swabs from Infected Cynomolgus Macaquesa Virus titre (log10 PFU/ml) of animals infected with: A/California/04/09 (H1N1) A/Kawasaki/UTK-4/09 (H1N1) Animal ID #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Nasal Day 1 5.8 1.0 1.5 4.7 4.5 2.6 3.0 2.9 1.6 3.6 2.4 3.6 swab Day 3 5.2 2.1 2.5 3.7 2.6 3.3 2.8 3.2 2.4 4.1 1.5 3.3 Day 5 4.7 4.6 3.4 1.3 3.6 2.5 —b Day 7 3.5 — — — 5.0 Tracheal Day 1 3.4 2.3 3.6 2.3 3.5 2.0 1.3 1.3 — 2.0 2.3 2.1 swab Day 3 4.3 — 2.6 2.6 2.4 2.0 1.0 1.8 — 4.0 — — Day 5 3.5 2.5 3.7 5.6 — — Day 7 — 2.0 — 3.4 — 2.6 Bronchial Day 1 2.9 2.4 3.7 2.2 3.3 — 1.5 — — — — — brush Day 3 3.5 — — 3.1 — — — 1.5 — — — — Day 5 4.4 2.4 1.8 4.4 — 1.3 Day 7 — — — 1.5 — 4.4 aCynomolgus macaques were inoculated with 10 7.4 PFU of virus (6.7 ml) through multiple routes. Nasal and tracheal swabs and bronchial brush samples were collected every other day for virus titration. b—. virus not detected (detection limit: 1.0 log PFU/ml). 10 Blank: not applicable, animals euthanized on day 3 pi.

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187 APPENDIX A TABLE A5-4 Virus Titres in Respiratory Organs of Infected Ferretsa Virus titres (mean log10 PFU ± SD/g) in: Virus Nasal turbinates Trachea Lungs 6.7+0.7 5.9+0.4 A/California/04/09 (H1N1) Day 3 3.53, 4.12 3.1+0.1 Day 6 2.40 2.95 7.3+0.6 6.0+1.7 A/Netherlands/603/09 (H1N1) Day 3 5.15 —b Day 6 3.26, 4.77 4.36, 5.14 7.8+0.6 6.0+0.9 A/Wisconsin/WSLH049/09 (H1N1) Day 3 6.49, 3.23 4.3+1.1 Day 6 4.57, 3.40 5.48 8.3+0.1 4.6+0.3 4.5+1.8 A/Wisconsin/WSLH34939/09 (H1N1) Day 3 4.5+1.0 3.8+1.4 3.6+1.1 Day 6 6.9+1.0 6.4+1.0 6.8+0.8 A/Osaka/164/09 (H1N1) Day 3 Day 6 — — — 6.5+0.5 A/Kawasaki/UTK-4/09 (H1N1) Day 3 2.45, 3.81 — Day 6 — 3.18 — aFerrets were intranasally infected with 10 6 PFU (500 μl) of virus. Three ferrets from each group were euthanized on days 3 and 6 pi for virus titration. When virus was recovered from all three animals, average titres are presented. When virus was not recovered from all three ferrets, individual titres were recorded. None of the viruses tested was recovered from the spleens, kidneys, brains, intestines, or livers of infected animals. b—. virus not detected (detection limit: 2.3 log PFU/g). 10

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188 IMPACTS OF THE 2009-H1N1 INFLUENZA A PANDEMIC TABLE A5-5 Virus Titres in Nasal Swabs of Inoculated and Contact Ferrets a Virus titres (mean log10 PFU/ml) in nasal swabs Virus Day 1 Day 3 Day 5 Day 7 Day 9 —b A/California/04/09 (H1N1) Pair 1 7.1 4.0 — — i — 6.8 4.3 — — c Pair 2 7.1 5.3 3.4 — — i — 6.2 4.0 — — c Pair 3 5.9 5.3 3.9 — — i — 6.4 5.9 2.1 — c A/Kawasaki/UTK-4/09 (H1N1) Pair 4 6.6 5.3 4.3 — — i — 5.0 4.5 2.5 — c Pair 5 6.1 5.9 1.3 — — i — — — — — c A/Victoria/03/75 (H3N2) Pair 6 6.3 3.9 2.0 — — i — — 6.0 3.7 2.5 c Pair 7 5.8 2.3 — — — i — — — — 2.3 c A/duck/Alberta/35/76 (H1N1) Pair 8 — 2.8 2.6 — — i — — — — — c Pair 9 — 2.3 — — — i — — — — — c Pair 10 — 4.9 3.7 4.0 — i — — — — — c pairs of ferrets, one animal was intranasally inoculated with 106 PFU of virus (500 μl) aFor (inoculated ferret, i) and one day later, a naïve ferret was placed in an adjacent cage (contact ferret, c). Nasal swabs were collected from inoculated and contact ferrets every other day for virus titration. b—. virus not detected (detection limit: 1.3 log PFU/ml). 10

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189 APPENDIX A TABLE A5-6 Virus Titres in Organs of Infected Miniature Pigsa Virus titres (log10 PFU/g) of infected animals with: A/California/04/09 (H1N1) A/swine/Hokkaido/2/81 (H1N1) Animal ID #1 #2 #5 #6 Nasal mucosa 6.7 5.0 5.1 4.8 Oro/nasopharynx 3.1 3.3 6.8 5.0 —b Tonsil 3.2 4.5 4.4 Trachea 6.3 5.5 5.8 5.3 Bronchus (right) 5.6 6.1 5.9 6.5 Bronchus (left) 6.7 6.5 5.4 6.3 Lung (upper right) 7.8 6.6 6.1 4.5 Lung (middle right) 7.5 6.7 6.1 5.5 Lung (lower right) 6.4 6.8 5.3 4.5 Lung (upper left) 6.8 6.4 6.8 5.1 Lung (middle left) 8.0 7.6 4.7 5.5 Lung (lower left) 6.2 7.4 5.5 4.7 Ileum — — 3.5 — Jejunum — — 2.8 — aSpecific-pathogen free miniature pigs were intranasally infected with 10 6.2 PFU (1 ml) of virus. Two animals from each group were euthanized on day 3 pi for virus titration. No virus was recovered from heart, spleen, kidneys, liver, duodenum, rectum, bladder, cerebrum, cerebellum, or brain stem. b—. virus not detected (detection limit: 2.0 log PFU/g). 10 TABLE A5-7 Virus Titres in Nasal Swabs from Infected Miniature Pigsa Virus titers (log10 PFU/ml) of infected animals with: A/California/04/09 (H1N1) A/swine/Hokkaido/2/81 (H1N1) Animal ID #1 #2 #3 #4 #5 #6 #7 #8 Day 1 5.6 6.5 6.4 6.1 6.3 5.3 3.6 4.1 Day 2 6.5 6.5 7.4 6.7 5.5 5.5 5.1 5.6 Day 3 5.7 5.3 7.2 5.3 5.0 4.4 4.7 5.5 Day 4 3.7 3.6 4.9 4.6 Day 5 4.5 5.4 2.7 3.2 Day 6 4.3 5.3 2.8 3.2 Day 7 3.3 3.4 1.3 1.6 —b Day 8 — — — Day 9 — — — — pigs were intranasally infected with 106.2 PFU of virus (1 ml) of virus. aMiniature b— .virus not detected (detection limit: 1.0 log10 PFU/ml). Blank: not applicable, animals euthanized on day 3 pi.

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TABLE A5-8 Virus Susceptibility to Antiviral Compounds in Cell Culture 190 IC90 A/California/04/09 A/Kawasaki/UTK-4/09 A/Kawasaki/UTK-23/08 (H1N1) (H1N1) (H1N1) Oseltamivir carboxylatea 10.56c 2971.30 5.58 Zanamivir 17.67 42.33 21.93 R-125489b 4.24 11.70 10.17 T-705 0.16 0.23 0.13 aOseltamivircarboxylate is the active form of oseltamivir. bR-125489 is the active form of CS-8958. cIC value: mean μg/ml or nM of triplicate reactions for T-705 and other compounds tested, respectively. 90 TABLE A5-9 Virus Sensitivity in Neuraminidase Assays IC50 A/California/04/09 A/Osaka/164/09 A/Kawasaki/UTK-4/09 A/Kawasaki/UTK-23/08 (H1N1) (H1N1) (H1N1) (H1N1) Oseltamivir carboxylatea 0.96c 1.6 1313 1.88 Zanamivir 0.32 0.43 0.79 0.36 R-125489b 0.41 0.44 0.34 0.20 aOseltamivircarboxylate is the active form of oseltamivir. bR-125489 is the active form of CS-8958. cIC value: mean nM of duplicate reactions. 50