A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets

Article

A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets Graphical Abstract

Authors High pathogenicity in mammals

Masaki Imai, Tokiko Watanabe, Maki Kiso, ..., James C. Paulson, Hideki Hasegawa, Yoshihiro Kawaoka

Correspondence

HPAI H7N9 viruses

[email protected] (M.I.), [email protected] (Y.K.)

In Brief Brain

Lung

Respiratory droplet transmission in ferrets Infected animals

Exposed animals

Highlights d

Highly pathogenic avian influenza (HPAI) H7N9 viruses replicate efficiently in mammals

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HPAI H7N9 viruses are more pathogenic than low pathogenic H7N9 viruses in mammals

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HPAI H7N9 viruses transmit via respiratory droplets among ferrets

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HPAI H7N9 viruses show low sensitivity to neuraminidase inhibitors in mice

Imai et al., 2017, Cell Host & Microbe 22, 1–12 November 8, 2017 ª 2017 Elsevier Inc. https://doi.org/10.1016/j.chom.2017.09.008

Highly pathogenic avian influenza (HPAI) H7N9 viruses have emerged and raised concerns of a pandemic. Imai et al. characterized an HPAI H7N9 virus isolated from a human. This virus transmitted among ferrets without prior adaptation and caused lethal infection in animals, demonstrating its pandemic potential and the need for surveillance.

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Cell Host & Microbe

Article A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets Masaki Imai,1,8,* Tokiko Watanabe,1,8 Maki Kiso,1,8 Noriko Nakajima,2,8 Seiya Yamayoshi,1,8 Kiyoko Iwatsuki-Horimoto,1,8 Masato Hatta,3,8 Shinya Yamada,1 Mutsumi Ito,1 Yuko Sakai-Tagawa,1 Masayuki Shirakura,4 Emi Takashita,4 Seiichiro Fujisaki,4 Ryan McBride,5 Andrew J. Thompson,5 Kenta Takahashi,2 Tadashi Maemura,1 Hiromichi Mitake,1 Shiho Chiba,3 Gongxun Zhong,3 Shufang Fan,3 Kohei Oishi,1 Atsuhiro Yasuhara,1 Kosuke Takada,1 Tomomi Nakao,1 Satoshi Fukuyama,1 Makoto Yamashita,1 Tiago J.S. Lopes,1,3 Gabriele Neumann,3 Takato Odagiri,4 Shinji Watanabe,4 Yuelong Shu,6 James C. Paulson,5 Hideki Hasegawa,2 and Yoshihiro Kawaoka1,3,7,9,* 1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan 2Department of Pathology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan 3Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Sciences, University of Wisconsin-Madison, Madison, WI 53711, USA 4Influenza Virus Research Center, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan 5Departments of Molecular Medicine & Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA 6National Institute for Viral Disease Control and Prevention, China Centers for Disease Control and Prevention, Beijing 102206, China 7Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan 8These authors contributed equally 9Lead Contact *Correspondence: [email protected] (M.I.), [email protected] (Y.K.) https://doi.org/10.1016/j.chom.2017.09.008

SUMMARY

Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-tomoderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.

INTRODUCTION Zoonotic influenza emergence in humans imposes a potentially huge public health and economic burden on a global level.

Low pathogenic avian influenza (LPAI) H7N9 viruses (which cause mild or asymptomatic disease in poultry) have caused five epidemic waves of human infection since they were first confirmed in China in 2013 (Shen and Lu, 2017; Wang et al., 2017; Zhou et al., 2017). The number of reported human cases increased appreciably during the fifth epidemic relative to the first four. As of July 26, 2017, there were 1,582 laboratoryconfirmed human cases of avian influenza H7N9 infection, with a fatality rate of approximately 39% (http://www.fao.org/ag/ againfo/programmes/en/empres/h7n9/situation_update.html). In February 2017, human infections with highly pathogenic avian influenza (HPAI) H7N9 viruses possessing a multi-basic cleavage site motif in hemagglutinin (HA) (which facilitates systemic virus replication in avian species) were first detected in Southern China during the fifth epidemic, and at least 25 cases were laboratory confirmed by July 2017 (http://www.fao.org/ag/againfo/ programmes/en/empres/h7n9/situation_update.html). Phylogenetic analysis indicated that the HPAI H7N9 viruses were derived from the LPAI H7N9 viruses currently circulating among domestic poultry (Ke et al., 2017; Zhang et al., 2017). The emergence of HPAI H7N9 viruses may represent an increased threat to human health, as the acquisition of a multi-basic cleavage site in HA is associated with increased pathogenicity in mammals (Hatta et al., 2001; Munster et al., 2010; Schrauwen et al., 2012; Suguitan et al., 2012). Sequence analysis of three human HPAI H7N9 isolates (A/Guangdong/17SF006/2017, A/Guangdong/17SF003/2016 [GD/3], and A/Taiwan/1/2017) revealed that A/Guangdong/17SF006/2017 and A/Taiwan/1/ 2017 possess the E627K mutation in the polymerase subunit PB2 protein (Ke et al., 2017), which confers efficient replication in mammals (Hatta et al., 2001; Subbarao et al., 1993), and that A/Taiwan/1/2017 has the K526R mutation in PB2, which

Cell Host & Microbe 22, 1–12, November 8, 2017 ª 2017 Elsevier Inc. 1

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

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Virus titer Log10 (PFU/ml)

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passaged the GD/3 isolate in embryonated chicken eggs to prepare a virus stock. Deep sequencing analysis of the virus stock revealed a mixed population of viruses encoding arginine at amino acid position 294 of NA (detected in 94% of sequence reads) or lysine (detected in 6% of sequence reads) (Table S1). Because GD/3 was isolated from an NA inhibitor-treated patient, the NA-294K variant may have emerged during the antiviral treatment of this patient. Although HPAI H7N9 viruses from the Guangdong environmental samples did not possess this mutation (Ke et al., 2017), the finding of an NA inhibitor-resistant subpopulation in a human H7N9 HPAI isolate is a major public health concern. We therefore created two recombinant viruses (rGD/3NA294R and rGD/3-NA294K, which possess arginine and lysine, respectively, at position 294 in NA in the background of the consensus sequence of the rest of GD/3), amplified them in Madin-Darby canine kidney cells, and characterized them together with GD/3 and an LPAI H7N9 virus (A/Anhui/1/2013; Anhui/1), which we (Watanabe et al., 2013) and others (Belser et al., 2013; Richard et al., 2013; Zhang et al., 2013) characterized previously.

Hours post-infection Figure 1. Growth Kinetics of Viruses in Differentiated NHBE Cells NHBE cells were infected with GD/3 (blue), rGD/3-NA294R (red), rGD/3NA294K (green), or Anhui/1 (gray) at an MOI of 0.001. The supernatants of the infected cells were harvested at the indicated times, and virus titers were determined by means of plaque assays in Madin-Darby canine kidney (MDCK) cells. Error bars indicate SDs from three independent experiments. The lower limit of detection is indicated by the horizontal dashed line.

contributes to enhance replication of H7N9 virus in combination with PB2-627K (Song et al., 2014). Moreover, the three human isolates encode the G186V mutation in HA (H3 numbering), which facilitates increased binding to human-type receptors (Dortmans et al., 2013; Ramos et al., 2013; Xiong et al., 2013b). Importantly, the three human isolates have an R294K substitution in the neuraminidase (NA) protein that confers resistance to NA inhibitors (McKimm-Breschkin et al., 1998). These findings suggest that further human adaptation may generate H7N9 strains with enhanced pathogenicity and transmissibility in humans. However, the replication capacity, pathogenicity, and transmissibility of human HPAI H7N9 isolates in mammals remain unknown. Here, we characterized a human HPAI H7N9 isolate and its recombinant derivatives in vitro and in vivo, and evaluated their pandemic potential. RESULTS Molecular Markers Associated with Pathogenicity, Replication, and Antiviral Resistance of A/Guangdong/ 17SF003/2016 (H7N9; GD/3) Virus Isolated from a Human To assess the pandemic potential of HPAI H7N9 viruses, we examined the biological properties of GD/3, an HPAI H7N9 virus isolated from a fatal human case treated with oseltamivir (Zhu et al., 2017). GD/3 does not encode the mammalian-adapting PB2-627K marker, but possesses an A588V substitution in PB2, which enhances H7N9 viral polymerase activity, replication in mammalian cells, and virulence in mice (Xiao et al., 2016). We 2 Cell Host & Microbe 22, 1–12, November 8, 2017

Growth Kinetics of HPAI H7N9 Viruses in Human Airway Epithelial Cells In differentiated normal human bronchial epithelial (NHBE) cells, rGD/3-NA294R grew to titers similar to those of Anhui/1 at 33
C and 37
C, temperatures corresponding to the upper and lower human airway, respectively (Figure 1). By contrast, rGD/3-NA294K showed delayed growth, as has been reported for other influenza viruses possessing this mutation (Yen et al., 2005). Interestingly, GD/3 also displayed reduced growth properties as 33
C. These results demonstrate that HPAI H7N9 viruses replicate efficiently in human airway epithelial cells, and that the NA-R294K mutation attenuates virus replication. Pathogenicity and Replication of HPAI H7N9 Viruses in Mice, Ferrets, and Cynomolgus Macaques Next, we evaluated the replication and pathogenicity of HPAI H7N9 viruses in mice, ferrets, and cynomolgus macaques (GD/3 only), which are well-established mammalian models for influenza virus research. In BALB/c mice, GD/3 and rGD/3NA294R were highly pathogenic with MLD50 (mouse lethal dose 50; the dose required to kill 50% of infected mice) values of 102.7 plaque-forming units (PFU) and 101.7 PFU, respectively (Figure 2). The rGD/3-NA294K virus displayed an MLD50 value of 104.8 PFU, comparable with that of the LPAI Anhui/1 virus (104.5 PFU), again demonstrating the attenuating effect of the NA294K mutation. LPAI Anhui/1 virus titers were higher on day 3 than on day 6 in the lungs and nasal turbinates of infected mice (Table S2). By contrast, virus titers of GD/3 and rGD/3NA294R remained high (lungs) or increased (nasal turbinates) on day 6 compared with day 3, while rGD/3-NA294K showed an intermediate phenotype. GD/3 replicated systemically with virus spread to the brain, whereas virus was not recovered from the brain of rGD/3-NA294R-, rGD/3-NA294K-, or Anhui/1-infected animals (Table S2). Sequencing analyses confirmed that the viruses in the lungs of rGD/3-NA294K-infected animals on day 6 post-infection retained lysine (K) at position 294 of NA. Together, these findings demonstrate the highly pathogenic

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Figure 2. Virulence in Mice Four mice per group were intranasally inoculated with 100, 101, 102, 103, 104, 105, or 106 PFU of GD/3, rGD/3-NA294R, rGD/3-NA294K, or Anhui/1. Survival (left panels) and body weight (right panels) were monitored daily. The values for body weights are means ± SD from live mice. See also Table S2.

nature of the HPAI H7N9 viruses in mice and the attenuating effect of the NA-R294K mutation. We next investigated the replication ability of GD/3, rGD/3NA294R, rGD/3-NA294K, and Anhui/1 in ferrets. On day 3 post-infection, similar titers were measured in nasal turbinates and trachea collected from ferrets, each infected with one of the four H7N9 viruses tested, although GD/3, rGD/3-NA294R, and Anhui/1 replicated in the lungs to higher titers than rGD/3NA294K (Figure 3A; Table S3). However, on day 6 post-infection, the mean virus titers of all three HPAI H7N9 viruses in the ferret lungs (mean titers = 5.3, 4.8, and 4.4 log10 [PFU/g], respectively) were higher than the mean virus titer of the LPAI Anhui/1 virus (mean titer = 1.9 log10 [PFU/g]), although these differences were not statistically significant for rGD/3-NA294K virus (Figure 3A; Table S3). Nonetheless, all four viruses exhibited relatively high levels of replication in the nasal turbinates at day 6 post-infection, consistent with our observations in mice infected with these viruses. Interestingly, all four viruses (including the LPAI Anhui/1 virus) were also recovered from the brains of two or three of the infected animals on day 6 post-infection; the finding of Anhui/1 virus replication in the brains of infected ferrets is consistent with our previous study (Watanabe et al., 2013). We

confirmed that the R294K mutation in NA was retained in the viruses recovered from the lungs of rGD/3-NA294K-infected animals on day 6 post-infection. Pathological examination of ferrets infected with the three HPAI H7N9 viruses revealed more severe lung lesions that extended across larger areas for ferrets infected with rGD/3-NA294R compared with those infected with GD/3 or rGD/3NA294K. Viral antigen-positive cells were detected in the trachea, bronchus, bronchial glands, and lungs of all ferrets infected with the three HPAI H7N9 viruses (Figures 3B–3D); however, more virus antigen-positive cells were detected in the lungs of animals infected with rGD/3NA294R. Viral antigens were also detected in the olfactory bulb, brain stem, and/or cerebral cortex of ferrets infected with GD/3, rGD/3-NA294R, or rGD/3NA294K at day 6 post-infection (Figure 3E), consistent with the notion that these viruses can replicate in the brain. Cynomolgus macaques (Macaca fascicularis) were infected with GD/3, resulting in slight weight loss (<6.8%) and transient fever in the absence of signs of severe disease (Figure S1), similar to infection with Anhui/1 in our previous study (Watanabe et al., 2013). Nonetheless, GD/3 replicated appreciably in the nasal turbinates, trachea, and lungs (Tables S4 and S5), although no virus was isolated from the nasal turbinates of one of three animals at 3 days post-infection. Overall, the virus titers and organ tropism of GD/3 are comparable with those of the LPAI Anhui/1 virus (Watanabe et al., 2013). Histopathological changes of the lungs revealed pulmonary edema, alveolar hemorrhage, and inflammatory cell infiltration in the alveolar spaces (Figure 4). Numerous viral antigen-positive cells were detected in the alveolar epithelial cells of all macaques. Virus antigens were also detected in the nasal turbinates, tonsils, duodenum (data not shown), and palpebral conjunctiva sections (Figure 4). GD/3 thus established a robust infection in the respiratory organs similar to our previous study with Anhui/1 (Watanabe et al., 2013). Respiratory Droplet Transmission of HPAI H7N9 Viruses in Ferrets Because efficient human-to-human transmission is a critical feature of pandemic influenza viruses, we examined the transmissibility of GD/3, rGD/3-NA294R, rGD/3-NA294K, and Anhui/1 Cell Host & Microbe 22, 1–12, November 8, 2017 3

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

(legend on next page)

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Figure 4. Histopathological Examination of the Lungs and Palpebral Conjunctiva of Infected Cynomolgus Macaques Representative pathological images of GD/3-infected lungs and palpebral conjunctiva on days 3 and 6 post-infection. Left panels, H&E staining. Right panels, immunohistochemistry for influenza viral antigen detection. Scale bars, 100 mm (lungs), 50 mm (palpebral conjunctiva). See also Figure S1 and Tables S4 and S5.

in a ferret model. In inoculated ferrets, no significant differences were observed among the nasal wash titers of GD/3, rGD/3NA294R, and Anhui/1 on days 1, 3, and 5 post-infection; how-

ever, rGD/3-NA294K titers were lower (Figure 5A). All infected animals experienced loss of appetite and lethargy, which was most severe for animals infected with rGD/3-NA294R. Importantly, one of the GD/3-infected, two of the rGD/3-NA294R-infected, and one of the rGD/3-NA294K-infected animals died on day 8, days 4 and 8, and day 6, respectively (Table S6); we did not observe lethality in our ferret replication study (see Figure 3), most likely because animals were killed on days 3 and 6 postinfection. Viruses were isolated from the respiratory organs and the brains of the dead animals, but not from any other organs (Table S6). To assess respiratory droplet transmissibility, we set up four transmission pairs, each comprising a naive ferret housed adjacent to an infected ferret 1 day post-infection, as described previously (Imai et al., 2012). We also compared the H7N9 viruses with A/California/04/2009 (CA04), a representative 2009 H1N1 pandemic virus. CA04 was transmitted to one out of two exposed ferrets (Figure 5A). Anhui/1 displayed limited transmissibility (one animal out of four), similar to previous studies (Belser et al., 2013; Richard et al., 2013; Watanabe et al., 2013). The HPAI H7N9 viruses were transmitted to one (GD/3), three (rGD/3-NA294R), or two (rGD/3NA294K) exposed animals. In the case of four pairs, it has been reported that only a combination of 0/4 and 4/4 indicates a significant difference (Nishiura et al., 2013). Therefore, the difference in transmission between each

Figure 3. Virus Replication and Pathological Findings in Infected Ferrets (A) Ferrets were infected intranasally with 106 PFU of virus. Three ferrets per group were euthanized on days 3 and 6 post-infection for virus titration. Virus titers in nasal turbinates, trachea, lung, and brain were determined by use of a plaque assay on MDCK cells. Horizontal bars show the mean (n = 3). Asterisks indicate significant differences in virus titers between compared viruses. *p < 0.05; **p < 0.01. (B–E) Shown are representative pathological findings in the (B) tracheae, (C) bronchus and bronchial glands, (D) lungs, (E) olfactory bulb, (E) brain stem, and (E) cerebral cortex of ferrets infected with the indicated viruses at 6 days post-infection with H&E staining (left panels) or with immunohistochemistry for influenza viral antigen detection (right panels). Scale bars, 50 mm (trachea, olfactory bulb, brain stem, and cerebral cortex), 100 mm (bronchus and bronchial glands, lung). See also Table S3.

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Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Figure 5. Respiratory Droplet Transmission among Ferrets and Postmortem Histopathological Examination of Brain and Respiratory Organs from Ferrets that Died during the Transmission Study (A) Ferrets were infected with 5 3 105 PFU of GD/3, rGD/3-NA294R, rGD/3-NA294K, Anhui/1, or CA04 (inoculated ferrets). One day later, four or two naive ferrets (exposed ferrets) were each placed in a cage adjacent to an infected ferret. Nasal washes were collected from infected ferrets on day 1 after inoculation and from exposed ferrets on day 1 after co-housing, and then every other day (for up to 15 days) for virus titration. (B) Histopathological lesions in the thalamus, brain stem, and cerebellum of ferrets (no. 10) that died on day 9 after exposure to rGD/3-NA294R. Scale bars, 50 mm. (C) Histopathological lesions in the tracheae, bronchus, and lungs of ferrets (no. 12) that died on day 6 after exposure to rGD/3-NA294R. Scale bars, 50 mm (trachea), 100 mm (bronchus, lung). Left panels, H&E staining. Right panels, immunohistochemistry for influenza viral antigen detection. See also Figure S2 and Tables S1, S6, and S7.

group in our study (the GD/3, rGD/3-NA294R, rGD/3-NA294K, and Anhui/1 groups were 1/4, 3/4, 2/4, and 1/4, respectively) was not statistically significant. Animals exposed to GD/3, rGD/3-NA294K, or Anhui/1 recovered from the infection, while two of three virus-positive animals exposed to rGD/3-NA294R succumbed to their infections on days 6 and 9 post-infection (Table S6); the inoculated ferrets in the respective transmission pairs also died on days 4 and 8, respectively. Virus was isolated from the respiratory organs and brains of the exposed animals that succumbed to their infec6 Cell Host & Microbe 22, 1–12, November 8, 2017

tions (Table S6). Given that ferrets infected with human or avian influenza viruses exhale infectious virus at a rate of less than 5 or 1 PFU per minute, respectively (Gustin et al., 2013), exposure via respiratory droplets to even a relatively small amount of this virus may be enough to cause lethal infection in ferrets. For the deceased exposed animals, we also performed histopathology and immunohistochemistry studies. Postmortem pathological examination demonstrated that appreciable numbers of neurons were positive for viral antigen and extensive inflammation was observed in the brain of one (ferret no. 10;

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Figure 5B) of the two dead animals. Viral antigens were also detected in the Purkinje cells. Although the virus replicated efficiently in the brain of ferret no. 12, no viral antigens were detected in the brain sections. In the lung lesions, epithelial detachment, inflammatory cell infiltration, edema, and hemorrhage were observed (Figure 5C). Seroconversion was detected in all exposed animals from which viruses were recovered (note, the two animals exposed to rGD/3-NA294R died before postexposure serum was collected; Table S7). Together, these results demonstrate that all three HPAI viruses (including the oseltamivir-resistant NA-294K variant) transmit via respiratory droplets among ferrets, and that rGD/3-NA294R caused lethal infections in inoculated and exposed animals. To identify amino acid changes that may support transmission via respiratory droplets among ferrets, we compared the consensus sequences of viruses isolated from infected and exposed animals. All viruses isolated from the nasal wash samples of ferrets infected with GD/3 encoded an N359S mutation in the polymerase protein PA, and a mixture of V219I/V in the M1 matrix protein (Figure S2); in fact, we detected these mutations in a small proportion of the viruses of the GD/3 virus stock (Table S1). The amino acid at position 219 of M1 is under selective pressure in humans (Furuse et al., 2009) and may play a role in influenza virus adaptation to mammals. Compared with the virus stocks used for infection, subpopulations of viruses isolated from nasal wash samples of the exposed ferret (no. 4) infected with GD/3 possessed two or four amino acid differences across four gene segments: one in PB2 (D701N), one in PA (N359S), one in NP (M191V), and one in M1 (R210K) (Figure S2). Of these four substitutions, PB2-D701N and PA-N359S were shared among GD/3 virus subpopulations from all four inoculated ferrets and from the infected exposed animal. The PB2-D701N mutation facilitates adaptation of avian influenza viruses to mammals (Li et al., 2005) and has been reported to contribute to transmission of H7N9 virus among ferrets (Zhu et al., 2015). However, the PB2-D701N and PA-N359S mutations were not detected in virus populations isolated from ferrets exposed to rGD/3-NA294R or rGD/3-NA294K, indicating that these two mutations are not necessary for HPAI H7N9 respiratory droplet transmissibility in ferrets. Although several other amino acid changes were detected in the infected and/or exposed animals, none was shared among all viral subpopulations. These findings demonstrate that the currently circulating HPAI H7N9 viruses can transmit via respiratory droplets among ferrets either as is or only with the mutations that are able to arise during a single round of infection and transmission. Receptor-Binding Specificity of HPAI H7N9 Viruses Since the currently circulating HPAI H7N9 viruses are transmitted among ferrets, we next tested their receptor-binding specificity, a known determinant of host-range and interspecies transmission (Neumann and Kawaoka, 2015). HAs from human strains preferentially bind to glycans terminating in sialic acids connected to galactose through an a2,6 linkage (‘‘humantype’’ receptors) on the cell surface; by contrast, the HAs of avian strains preferentially bind to sialic acids linked to galactose by an a2-3-linkage (‘‘avian-type’’ receptors) (Rogers and Paulson, 1983). The HA of GD/3 possesses mutations that were found to be associated with increased binding to human-type recep-

tors in an LPAI H7N9 isolate, namely S138A, G186V, and T221P (Shi et al., 2013). To characterize the receptor-binding properties of the viruses tested here, we used a glycan array in which 129 diverse sialic acid-containing glycans were printed on a microarray (Table S8). Recombinant viruses possessing the HA and NA genes of GD/3, Anhui/1, or A/Kawasaki/173/ 2001 (H1N1; K173; chosen because of its known human-type receptor-binding specificity) (Imai et al., 2012), and the remaining genes from A/Puerto Rico/8/34 (H1N1; PR8) (Ping et al., 2015) were generated by reverse genetics, and subjected to glycan array analysis. K173 virus bound preferentially to a2,6-linked glycans (Figure 6A), as expected. Anhui/1 (carrying HA-226L, which is commonly found in seasonal human influenza viruses) exhibited appreciable binding to N-linked a2,6-linked sialosides, whereas GD/3 (carrying HA-226Q, which is commonly found in avian viruses) demonstrated binding specificity for a2,3-linked sialosides. To further characterize the receptor-binding properties of the H7N9 viruses, we performed binding assays using biolayer interferometry (Figure 6B). K173 preferentially bound to a2,6-linked glycans (6SLN, 6SLNLN, and 6SLNLNLN). As reported elsewhere (Stevens et al., 2006, 2008), A/Vietnam/1203/2004 (H5N1; VN1203), which served as a control virus with typical avian-type receptor specificity, bound to only an a2,3-linked glycan (3SLN). Anhui/1 bound to a2,3- and a2,6-linked glycans, which is consistent with the results of glycan array analysis. Unlike the results of the glycan array analysis, however, GD/3 showed dual a2,3- and a2,6-linked glycan receptor specificity. This finding is consistent with a previous report of a GD/3 isolate recognizing both avian- and human-type receptors in an ELISA in which a2,3- or a2,6-linked sialylglycopolymers were coated onto a microtiter plate (Zhu et al., 2017). The reason for this discrepancy is unclear; however, it might be due to differences in the antibodies, virus preparations, and/or experimental settings (for example, biolayer interferometry and ELISA may detect weak binding to human-type receptors, whereas the glycan microarray is a more stringent test of specificity; Chen et al., 2012). Accordingly, GD/3 recognizes human-type receptors to some extent, even though GD/3 binding to a2,6-linked sialosides was not detected in our glycan array analysis. Antiviral Susceptibility of HPAI H7N9 Viruses Antiviral compounds are currently the only option for the treatment and prophylaxis of H7N9 human infections. To assess whether current anti-influenza drugs are effective against HPAI H7N9 viruses, we determined the in vitro half maximal inhibitory concentration of several NA inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir), and of an inhibitor of the viral RNA polymerase (favipiravir, also known as T-705) against GD/3, rGD/3-NA294R, and rGD/3-NA294K. GD/3 and rGD/3-NA294R were sensitive to all NA inhibitors tested (Table S9), demonstrating that the minor subpopulation of the NA inhibitor-resistant NA294K mutant in the GD/3 virus stock did not appreciably affect virus sensitivity to NA inhibitors (Table S1). In contrast, rGD/3-NA294K was resistant to NA inhibitors, as reported recently by Zhu et al. (2017). Similar results were obtained with the NA inhibitor-sensitive Shanghai/1-NA-294R and the drugresistant Shanghai/1-NA-294K strains, which served as controls (Watanabe et al., 2013). The polymerase inhibitor favipiravir was Cell Host & Microbe 22, 1–12, November 8, 2017 7

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

A

B

Figure 6. Virus Binding to Human and Avian Receptors (A) The receptor specificities of two recombinant viruses possessing H7N9 virus HAs (GD/3 and Anhui/1) were compared with a representative human (K173) isolate by using a glycan microarray containing a diverse library of a2-3 and a2-6 sialosides. Viruses were applied at 128–256 hemagglutination units/ml for 1 hr, and after washing, viruses were detected with monoclonal anti-H7 mouse immunoglobulin G (IgG) (for H7N9 viruses) or anti-H1 mouse IgG (for the human K173 virus) and Alexa Fluor 488-labeled anti-mouse IgG secondary antibodies. Error bars represent the SD calculated from six replicate spots of each glycan. A complete list of glycans is provided in Table S8. (B) Virus binding to a2,3- and a2,6-linked glycans was determined by biolayer interferometry. Streptavidin biosensors were immobilized with a2,3-linked (3SLN) or a2,6-linked (6SLN, 6SLNLN, and 6SLNLNLN) sialylglycan receptors and reacted with replication-incompetent virus in the presence of NA inhibitors for 4,000 s at 30
C. Blue squares, N-acetylglucosamine; yellow circles, galactose; purple diamonds, sialic acid.

active against all viruses tested (Table S10), suggesting that it may be an effective treatment option against NA inhibitor-resistant HPAI H7N9 viruses. After establishing the in vitro sensitivity to antiviral compounds, we also assessed the therapeutic efficiency of the anti-influenza drugs in mice infected with GD/3, rGD/3NA294R, rGD/3-NA294K, or Anhui/1. Peramivir, which is structurally similar to oseltamivir but administered intravenously, was omitted from these experiments. Mice infected with 103 PFU of viruses were treated with the drugs beginning 2 hr post-infection. Under the conditions tested here, the NA inhibitors had only limited effects on body weight loss and lung virus titers (Figures 7 and S3). When rGD/3-NA294K-infected mice were treated with the NA inhibitors, however, no differences in weight loss or viral titers were observed, likely due to the reduced susceptibility of rGD/3-NA294K to NA inhibitors. By contrast, favipiravir, which targets the viral polymerase com8 Cell Host & Microbe 22, 1–12, November 8, 2017

plex, showed clear therapeutic effectiveness against all four viruses. DISCUSSION Our study shows that a human HPAI H7N9 isolate (GD/3) and two recombinant viruses (rGD/3-NA294R and rGD/3-NA294K, which differ in their susceptibility to NA inhibitors) replicated efficiently in the upper and lower respiratory tracts of mice, ferrets, and/or nonhuman primates, and were more pathogenic in both mice and ferrets than an LPAI H7N9 virus (Anhui/1), with the exception of the NA inhibitor-resistant virus (rGD/3-NA294K). Notably, the three HPAI viruses exhibited more robust replication in the brain of ferrets, and caused lethal infections in contrast to the LPAI virus. Moreover, it is noteworthy that two of the three exposed ferrets infected with the oseltamivir-sensitive NA-294R variant succumbed to their infections, and one experienced severe

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

A

B

Figure 7. Virus Sensitivity to Antivirals in Mice Six mice per group were intranasally inoculated with 103 PFU (50 mL) of GD/3, rGD/3-NA294R, rGD/3-NA294K, or Anhui/1. At 2 hr after infection, mice were treated with the antiviral compounds shown. (A) Treatment of infected mice with laninamivir (administered once) compared with oseltamivir or favipiravir (administered daily). Mice were treated with: (1) 40 mg/kg per 200 mL of oseltamivir phosphate orally twice daily for 5 days after infection; (2) 60 or 150 mg/kg per 200 mL of favipiravir orally twice daily for 5 days after infection; or (3) 0.75 mg/kg per 50 mL of laninamivir intranasally once on the day of infection. (B) Comparison of the efficacy of daily-administered zaninamivir with that of favipiravir. Mice were treated with: (1) 8 mg/kg per 50 mL of zanamivir intranasally once daily for 5 days post-infection; (2) 60 or 150 mg/kg per 200 mL of favipiravir orally twice daily for 5 days post-infection. Saline (50 mL) or methylcellulose (200 mL) served as controls for intranasal or oral treatment, respectively. The detailed experimental design is shown in Figure S3. Body weights were monitored daily (left panels). For virological examinations, three mice per group were euthanized at 3 and 6 days post-infection and the virus titers in the lungs were determined by means of plaque assays in MDCK cells (right panels). Statistically significant differences between virus titers of control mice and those of mice treated with antiviral drugs were determined using one-way ANOVA, followed by Dunnett’s test. *p < 0.05. Error bars denote SDs. See also Tables S9 and S10.

encephalitis. Given that the amount of virus needed to initiate infection of exposed ferrets via respiratory droplets is small, these findings suggest that the HPAI H7N9 virus requires only a limited amount of virus to cause lethal infection at least in this animal model. The fact that respiratory droplet-exposed ferrets succumbed to their infections clearly suggests a substantial increase in the pathogenicity of HPAI H7N9 viruses in mammals compared with their progenitor LPAI H7N9 viruses, requiring close monitoring of HPAI H7N9 viruses in the field. We observed that ferrets inoculated with the HPAI H7N9 viruses exhibited delayed viral clearance from the lungs compared with the LPAI H7N9-infected ferrets, with the exception of the NA inhibitor-resistant virus (Figure 3A). In addition, the most severe histopathological changes were found in the lungs of animals in-

fected with the oseltamivir-sensitive NA-294R variant, indicating that the extensive inflammatory cell infiltration contributed to the severity of the lung injury. Thus, the prolonged virus clearance also seemed to contribute to the severity of disease in these ferrets. HA receptor-binding specificity is known to be a key determinant of efficient transmission among mammals (Neumann and Kawaoka, 2015). In this study, we found that all three HPAI viruses tested (GD/3, rGD/3-NA294R, and rGD/3-NA294K) transmitted via respiratory droplets among ferrets, although they possessed avian-type receptor-binding specificity in our glycan microarray assay (Figure 6A). However, we observed that GD/3 can recognize both avian- and human-type receptors by using a biolayer interferometry assay (Figure 6B). In addition, a recent Cell Host & Microbe 22, 1–12, November 8, 2017 9

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

study reported the binding of a GD/3 isolate to both avian- and human-type receptors (Zhu et al., 2017). Studies using the ferret and guinea pig models indicate that virus transmission capability does not depend solely on receptor specificity, but is a polygenic trait (Neumann and Kawaoka, 2015). Relatively efficient binding to host cells is most likely critical, whereas other viral components may affect the efficiency of virus transmission among ferrets. A human HPAI H7N9 isolate (GD/3) used in this study exhibited lethal pathogenicity in both mice and ferrets, but moderate pathogenicity in cynomolgus macaques. The pathogenicity of influenza viruses has been shown to vary depending on the animal model and/or strain of influenza virus used. For example, some HPAI H5N1 strains isolated from humans are not lethal in nonhuman primates (Muramoto et al., 2014), whereas the same strains cause lethal systemic infections in mice (Le et al., 2010). Our recombinant HPAI H7N9 virus encoding NA-294R (i.e., rGD/3-NA294R) was susceptible to NA inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir) in vitro. However, we found that treatment with NA inhibitors had little-to-no effect on body weight loss and/or lung virus titers in mice infected with the NA inhibitor-sensitive virus (Figure 7). Similar findings were obtained with LPAI H7N9 and 2009 pandemic H1N1 viruses; mice infected with NA inhibitor-sensitive variants of these viruses and treated with NA inhibitors showed body weight loss (Ranadheera et al., 2016; Watanabe et al., 2013). Thus, the NA inhibitors may not provide effective treatment against the NA inhibitor-sensitive viruses of some influenza A virus subtypes in the mouse model. A number of studies have reported that NA mutations associated with resistance to NA inhibitors compromise viral fitness in vitro and in vivo (Baranovich et al., 2011). We also observed that a recombinant HPAI H7N9 virus with R294K, generated on the basis of the consensus sequence of the GD/3 virus stock, was attenuated in differentiated NHBE cells, mice, and ferrets relative to its NA inhibitor-sensitive counterpart, suggesting that the reduction in replicative fitness of HPAI H7N9 viruses is caused by the R294K mutation. Nevertheless, HPAI H7N9 viruses carrying the R294K mutation have been isolated from patients with high frequency (8 out of 30 human isolates; http://platform.gisaid.org/epi3/frontend#5541a). A compensatory mutation(s) that restores the viral replication fitness of the oseltamivir-resistant mutant of HPAI H7N9 viruses might be present in the virus populations recovered from patients treated with oseltamivir. HPAI viruses of the H5N1 subtype have caused a substantial number of human infections in Southeast Asia and Egypt, raising concern of a pandemic threat. To date, no sustained transmission of H7N9 or H5N1 viruses among humans has been documented. However, further adaptation of these avian viruses to humans may result in transmissible viruses with pandemic potential. Studies in ferrets have demonstrated that HPAI H5N1 viruses require several mutations to transmit efficiently via respiratory droplets (Chen et al., 2012; Herfst et al., 2012; Imai et al., 2012; Jackson et al., 2009; Maines et al., 2006). Our findings showed that HPAI H7N9 viruses possess the ability to transmit among ferrets without prior adaptation. Importantly, these viruses can cause severe disease, which can lead to fatal 10 Cell Host & Microbe 22, 1–12, November 8, 2017

outcomes. Moreover, the drug-resistant NA-294K variant has retained its pathogenicity and transmissibility in ferrets. Collectively, these data suggest that HPAI H7N9 viruses may be closer to acquiring efficient transmission in humans, and, therefore, have greater pandemic potential than the HPAI H5N1 viruses. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell Cultures B Viruses B Mice B Ferrets B Cynomolgus Macaques METHOD DETAILS B Antiviral Compounds B Reverse Genetics B Generation of Replication-Defective Virus B Deep Sequencing B DNA Sequencing B Growth Kinetics of Virus in Cell Culture B Hemagglutination Inhibition (HI) Assay B Animal Experiments B Experimental Infection of Mice B Experimental Infection of Ferrets B Ferret Transmission Study B Experimental Infection of Cynomolgus Macaques B Pathological Examination B NA Inhibition Assay B Plaque Reduction Assay B Antiviral Sensitivity of Viruses in Mice B Glycan Arrays B Biolayer Interferometry QUANTIFICATION AND STATISTICAL ANALYSIS B Biosafety Statement

SUPPLEMENTAL INFORMATION Supplemental Information includes 3 figures and 11 tables and can be found with this article online at https://doi.org/10.1016/j.chom.2017.09.008. AUTHOR CONTRIBUTIONS M. Imai, T.W., M.K., N.N., S. Yamayoshi, K.I.-H., M.H., S. Yamada, M. Ito, Y.S.-T., M.S., E.T., S. Fujisaki, R.M., A.J.T., K. Takahashi, T.M., H.M., S.C., G.Z., S. Fan, K.O., A.Y., K. Takada, T.N., S. Fukuyama, M.Y., S.W., and H.H. performed the experiments. M. Imai, T.W., M.K., N.N., S. Yamayoshi, K.I.-H., M.H., A.J.T., S. Fukuyama, M.Y., T.J.S.L., G.N., T.O., S.W., Y.S., J.C.P., H.H., and Y.K. planned the experiments and/or analyzed the data. M. Imai, T.W., M.K., N.N., S. Yamayoshi, K.I.-H., M.H., G.N., T.O., S.W., Y.S., J.C.P., H.H., and Y.K. wrote the manuscript. Y.K. oversaw the project. ACKNOWLEDGMENTS We thank the Global Initiative on Sharing Avian Influenza Data (GISAID) for sharing the influenza virus sequence data used in this study. We thank Toyama Chemical Co., Ltd, for providing favipiravir, and Daiichi Sankyo Inc. for

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

providing laminamivir. We thank Susan Watson for scientific editing. We also thank Naomi Fujimoto, Mikiko Tanaka, Naoko Midorikawa, Takeaki Imamura, Yuko Sato, Kelly Moore, Alexander Karasin, and David Hinkel for technical assistance. This research was supported by Leading Advanced Projects for medical innovation (LEAP) from the Japan Agency for Medical Research and Development (AMED), by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan (nos. 16H06429, 16K21723, and 16H06434), by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from AMED, by e-ASIA Joint Research Program from AMED, by the Research Program on Emerging and Re-emerging Infectious Diseases from AMED, and by the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C). M.Y. has received grant support from Daiichi Sankyo Co., Ltd. G.N. is a co-founder of FluGen. Y.K. has received grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, and Ttsumura Co., Ltd, royalties from MedImmune, and is a co-founder of FluGen. Received: June 29, 2017 Revised: August 3, 2017 Accepted: September 15, 2017 Published: October 19, 2017 REFERENCES Baranovich, T., Webster, R.G., and Govorkova, E.A. (2011). Fitness of neuraminidase inhibitor-resistant influenza A viruses. Curr. Opin. Virol. 1, 574–581. Belser, J.A., Gustin, K.M., Pearce, M.B., Maines, T.R., Zeng, H., Pappas, C., Sun, X., Carney, P.J., Villanueva, J.M., Stevens, J., et al. (2013). Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature 501, 556–559. Chen, L.M., Blixt, O., Stevens, J., Lipatov, A.S., Davis, C.T., Collins, B.E., Cox, N.J., Paulson, J.C., and Donis, R.O. (2012). In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology 422, 105–113. Dortmans, J.C., Dekkers, J., Wickramasinghe, I.N., Verheije, M.H., Rottier, P.J., van Kuppeveld, F.J., de Vries, E., and de Haan, C.A. (2013). Adaptation of novel H7N9 influenza A virus to human receptors. Sci. Rep. 3, 3058. Furuse, Y., Suzuki, A., Kamigaki, T., and Oshitani, H. (2009). Evolution of the M gene of the influenza A virus in different host species: large-scale sequence analysis. Virol. J. 6, 67. Gustin, K.M., Katz, J.M., Tumpey, T.M., and Maines, T.R. (2013). Comparison of the levels of infectious virus in respirable aerosols exhaled by ferrets infected with influenza viruses exhibiting diverse transmissibility phenotypes. J. Virol. 87, 7864–7873. Hatta, M., Gao, P., Halfmann, P., and Kawaoka, Y. (2001). Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842. Herfst, S., Schrauwen, E.J., Linster, M., Chutinimitkul, S., de Wit, E., Munster, V.J., Sorrell, E.M., Bestebroer, T.M., Burke, D.F., Smith, D.J., et al. (2012). Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336, 1534–1541. Hothorn, T., Bretz, F., and Westfall, P. (2008). Simultaneous inference in general parametric models. Biom. J. 50, 346–363. Imai, M., Watanabe, T., Hatta, M., Das, S.C., Ozawa, M., Shinya, K., Zhong, G., Hanson, A., Katsura, H., Watanabe, S., et al. (2012). Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486, 420–428. Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., Tamura, D., Sakai-Tagawa, Y., Noda, T., et al. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 460, 1021–1025. Iwasaki, T., Tamura, S., Kumasaka, T., Sato, Y., Hasegawa, H., Asanuma, H., Aizawa, S., Yanagihara, R., and Kurata, T. (1999). Exacerbation of influenzavirus pneumonia by intranasal administration of surfactant in a mouse model. Arch. Virol. 144, 675–685.

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Zhu, W., Li, L., Yan, Z., Gan, T., Li, L., Chen, R., Chen, R., Zheng, Z., Hong, W., Wang, J., et al. (2015). Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models. Sci. Rep. 5, 14170.

Suguitan, A.L., Jr., Matsuoka, Y., Lau, Y.F., Santos, C.P., Vogel, L., Cheng, L.I., Orandle, M., and Subbarao, K. (2012). The multibasic cleavage site of the hemagglutinin of highly pathogenic A/Vietnam/1203/2004 (H5N1) avian influenza virus acts as a virulence factor in a host-specific manner in mammals. J. Virol. 86, 2706–2714.

Zhu, W., Zhou, J., Li, Z., Yang, L., Li, X., Huang, W., Zou, S., Chen, W., Wei, H., Tang, J., et al. (2017). Biological characterisation of the emerged highly pathogenic avian influenza (HPAI) A(H7N9) viruses in humans, in mainland China, 2016 to 2017. Euro Surveill. 22, https://doi.org/10.2807/1560-7917.ES.2017. 22.19.30533.

12 Cell Host & Microbe 22, 1–12, November 8, 2017

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal antibody for type A influenza nucleoprotein antigen

Iwasaki et al., 1999

N/A

Mouse monoclonal anti-H7 antibody

This study

N/A

Mouse monoclonal anti-H1 antibody

Abcam

Cat#ab128412; RRID: AB_11142609

Anti-mouse Alexa fluor-488-conjugated secondary antibody

Thermo Fisher Scientific

Cat#A-11029; RRID: AB_138404

N/A

Antibodies

Bacterial and Virus Strains A/Guangdong/17SF003/2016 (H7N9) virus

Zhu et al., 2017

A/Anhui/1/2013 (H7N9) virus

Watanabe et al., 2013

N/A

A/California/04/2009 (H1N1pdm) virus

Itoh et al., 2009

N/A

Plaque-purified A/Shanghai/1/2013 (H7N9) virus with NA294K

Watanabe et al., 2013

N/A

Plaque-purified A/Shanghai/1/2013 (H7N9) virus with NA294R

Watanabe et al., 2013

N/A

Recombinant virus: rGD/3-NA294R

This study

N/A

Recombinant virus: rGD/3-NA294K

This study

N/A

Recombinant virus possessing the HA and NA genes of GD/3, and the remaining genes from PR8

This study

N/A

Recombinant virus possessing the HA and NA genes of Anhui/1, and the remaining genes from PR8

This study

N/A

Recombinant virus possessing the HA and NA genes of K173, and the remaining genes from PR8

Imai et al., 2012

N/A

Replication-incompetent viruses expressing EGFP from the PB2 gene of PR8

This study

N/A

Laninamivir

Daiichi Sankyo

N/A

Laninamivir octanoate

Daiichi Sankyo

N/A

Favipiravir

Toyama Chemical

N/A

Oseltamivir carboxylate

F. Hoffmann-La Roche

N/A

Zanamivir

GlaxoSmithKline

N/A

Peramivir

Sequoia Research Products

N/A

Methylcellulose

Wako Pure Chemical Industries

Cat#133-17815

Superscript III reverse transcriptase

Thermo Fisher Scientific

Cat#18080044

b-propiolactone

Sigma-Aldrich

Cat#P5648

3SLN

Tokyo Chemical Industry

N/A

6SLN

Tokyo Chemical Industry

N/A

6SLNLN

Tokyo Chemical Industry

N/A

6SLNLNLN

Tokyo Chemical Industry

N/A

NEBNext dsDNA Fragmentase

New England BioLabs

Cat#M0348L

Blasticidin

InvivoGen

Cat#ant-bl-1

Chemicals, Peptides, and Recombinant Proteins

Critical Commercial Assays QIAamp Viral RNA Mini kit

Qiagen

Cat#52906

One-Step SuperScript III RT-PCR kit

Thermo Fisher Scientific

Cat#12574035 (Continued on next page)

Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017 e1

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Agencourt AMPure XP

Beckman Coulter

Cat#A63882

NEBNext Ultra II DNA Library Prep kit for Illumina

New England BioLabs

Cat#E7645L

MiSeq Reagent Kit v3 150-cycle

Illumina

Cat#MS-102-3001

BigDye Terminator version 3.1 Cycle Sequencing Kits

Thermo Fisher Scientific

Cat#433745

Biosensor / Streptavidin (SA) Tray

ForteBio

Cat#18-5019

NA-Fluor Influenza Neuraminidase Assay Kit

Thermo Fisher Scientific

Cat#4457091

Dako EnVision+ System-HRP Labelled Polymer Anti-Rabbit

Agilent

Cat#K4003

Experimental Models: Cell Lines MDCK; Madin-Darby canine kidney

N/A

N/A

293T; human embryonic kidney

N/A

N/A

NHBE; Human Bronchial/Tracheal Epithelial Cells

Lonza

Cat#CC-2540

MDCK cells stably expressing the PB2 protein derived from A/Puerto Rico/8/34 strain

Ozawa et al., 2011

N/A

Mice: BALB/c

Japan SLC inc.

N/A

Ferrets

Triple F Farms

N/A

Cynomolgus macaques

Shin Nippon Biomedical Laboratories

N/A

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-PB2

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-PB1

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-PA

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-HA

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-NP

This Study

N/A

pHH21-A/Guangdong/17SF003/2016NA-294K

This Study

N/A

pHH21-A/Guangdong/17SF003/2016NA-294R

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-M

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-NS

This Study

N/A

pHH21-A/Guangdong/17SF003/2016-delHA

This Study

N/A

pHH21-Anhui-HA

This Study

N/A

Experimental Models: Organisms/Strains

Oligonucleotides See Table S11 Recombinant DNA

pHH21-Anhui-NA

This Study

N/A

pPolI-VN1203 HA R1

This Study

N/A

pPolI-VN1203 NA

This Study

N/A

pPolI-K173-HA

This Study

N/A

pPolI-K173-NA

This Study

N/A

pPolI-PB2(120)EGFP(336)

Kobayashi et al., 2013

N/A

pPolI-PR8-HY/PB2(UW-PB2 C4U I504V)

Ping et al., 2015

N/A

pPolI-PR8-HY/PB1(UW-PB1 C4U M40L/G180W)

Ping et al., 2015

N/A

pPolI-PR8-HY/PA(UW-PA C4U R401K)

Ping et al., 2015

N/A

pPolI-PR8-HY/NP(UW-NP I116L)

Ping et al., 2015

N/A

pPolI-PR8-HY/M

Ping et al., 2015

N/A

pPolI-PR8-HY/NS(UW-NS1 A30P/R118K)

Ping et al., 2015

N/A (Continued on next page)

e2 Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

pCAGGS-WSN-PB2

Neumann et al., 1999

N/A

pCAGGS-WSN-PB1

Neumann et al., 1999

N/A

pCAGGS-WSN-PA

Neumann et al., 1999

N/A

pCAGGS-WSN-NP

Neumann et al., 1999

N/A

pCAGGS-PR8-PB2

Ping et al., 2015

N/A

pCAGGS-PR8-PB1

Ping et al., 2015

N/A

pCAGGS-PR8-PA

Ping et al., 2015

N/A

pCAGGS-PR8-NP

Ping et al., 2015

N/A

GraphPad Prism

GraphPad

https://www.graphpad.com

R

The R Foundation

https://www.r-project.org

CLC Genomics Workbench version 7.5.5

QIAGEN

https://www.qiagenbioinformatics.com/ products/clc-genomics-workbench/ Cat#11430-030

Software and Algorithms

Other Eagle’s minimal essential medium (MEM)

Thermo Fisher Scientific

Dulbecco’s modified Eagle’s medium (DMEM)

Sigma-Aldrich

Cat#D5796

Newborn calf serum (NCS)

Thermo Fisher Scientific

Cat#16010159

Fetal calf serum

EQUITECH-BIO

Cat#268-1

Chicken red blood cells

NIPPON BIO-TEST LABORATORIES

Cat#0109-1

Turkey red blood cells

NIPPON BIO-TEST LABORATORIES

N/A

Glycan array

Peng et al., 2017

N/A

HBS-EP Buffer

GE Healthcare Life Sciences

Cat#BR100188

TPCK-treated trypsin

Worthington Biochemical

Cat#LS03744

Bovine serum albumin

Sigma-Aldrich

Cat#A8412

10% phosphate-buffered formalin

Wako Pure Chemical Industries

Cat#060-01667

PBS

Sigma-Aldrich

Cat#D1408

RDE II (Receptor Destroying Enzyme)

Denka Seiken

Cat#370013

TransIT-293 Transfection Reagent

Mirus

Cat#MIR 2706

TRIzol LS Reagent

Thermo Fisher Scientific

Cat#10296028

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yoshihiro Kawaoka ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Cultures MDCK cells were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum (NCS). Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Normal human bronchial epithelial cells (NHBEs) were obtained from Lonza. The NHBE cell monolayers were cultured and differentiated as previously described (Itoh et al., 2009). MDCK cells stably expressing the PB2 protein derived from A/Puerto Rico/8/34 (H1N1; PR8) were maintained in 5% NCS/MEM containing blasticidin (10 mg/ml) (Ozawa et al., 2011). All cells were incubated at 37
C with 5% CO2. Viruses GD/3 (Zhu et al., 2017), Anhui/1 (Watanabe et al., 2013), and A/Shanghai/1/2013 (H7N9) (Watanabe et al., 2013) viruses were propagated in embryonated chicken eggs. CA04 (Itoh et al., 2009) was propagated in MDCK cells. All experiments with H7N9 viruses were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo and the National Institute of Infectious Diseases, Japan, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan, or in enhanced BSL3 containment laboratories at the University of Wisconsin-Madison, which are approved for such use by the Centers for Disease Control and Prevention and by the US Department of Agriculture. Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017 e3

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Mice Five-week-old female BALB/c mice were purchased from Japan SLC Inc., and housed in individually ventilated cages (Isocages; Techniplast) in enhanced BSL3 containment laboratories at the University of Tokyo. The mice were 6 weeks old at the time of infection. All experiments with mice were performed in accordance with the University of Tokyo’s Regulations for Animal Care and Use and approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. Ferrets Six-month-old female ferrets were obtained from Triple F Farms, and housed in individually ventilated cages (Showa Science) in enhanced BSL3 containment laboratories at the University of Tokyo. The ferrets were 6 to 8 months old at the time of infection. All experiments with ferrets were performed in accordance with the University of Tokyo’s Regulations for Animal Care and Use and approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. Cynomolgus Macaques Approximately 3-year-old male cynomolgus macaques (Macaca fascicularis) from Cambodia were purchased from Shin Nippon Biomedical Laboratories, and housed in individually ventilated cages in enhanced BSL3 containment laboratories at the Shin Nippon Biomedical Laboratories. All experiments with macaques were performed in accordance with the animal welfare bylaws of Shin Nippon Biomedical Laboratories, Drug Safety Research Laboratories, which is fully accredited by AAALAC International, and were approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories. METHOD DETAILS Antiviral Compounds Laninamivir and laninamivir octanoate were kindly provided by Daiichi Sankyo. Favipiravir was kindly provided by Toyama Chemical and oseltamivir carboxylate was kindly provided by F. Hoffmann-La Roche. Zanamivir was kindly provided by GlaxoSmithKline. Peramivir was obtained from Sequoia Research Products. For the mouse studies, oseltamivir phosphate and favipiravir were diluted in methylcellulose, whereas laninamivir and zanamivir were diluted in sterile saline. Reverse Genetics Plasmid-based reverse genetics for influenza virus generation was performed as previously described (Neumann et al., 1999). In brief, plasmids encoding the complementary DNAs for the eight viral RNA segments under the control of the human RNA polymerase I promoter and the mouse RNA polymerase I terminator (referred to as PolI plasmids), and plasmids for the expression of the viral PB2, PB1, PA and nucleoprotein proteins derived from a laboratory-adapted influenza A virus strain A/WSN/33 (H1N1), under the control of the chicken b-actin promoter (Niwa et al., 1991), were transfected into 293T cells with the help of a transfection reagent, Trans-IT 293 (Mirus). At 48 h post-transfection, culture supernatants were collected and inoculated to MDCK cells for virus propagation. All virus stocks were sequenced to confirm the absence of unwanted mutations. Generation of Replication-Defective Virus Replication-incompetent viruses expressing enhanced green fluorescent protein (EGFP) from the PB2 gene of PR8 were generated by using reverse genetics as described previously (Neumann et al., 1999). Plasmid constructs for viral RNA production (pPolI)—containing the genes of the GD/3, Anhui/1, PR8, K173, and VN1203 viruses flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—were constructed as described elsewhere (Neumann et al., 1999). A plasmid [pPolIPB2(120) EGFP(336)] was constructed to replace the PolI plasmid encoding the PB2 segment and contained the PR8-derived 30 PB2 noncoding region, 120 nucleotides that correspond to the PB2-coding sequence at the 30 end of the vRNA followed by the EGFP-coding sequence, 120 nucleotides that correspond to the PB2-coding sequence at the 50 end of the vRNA, and finally the 50 PB2 noncoding region (Kobayashi et al., 2013). The multibasic amino acids at the HA cleavage site (EVPKRKRTARYG or RERRRKKRYG) of GD/3 or VN1203 were changed to EIPKGRYG or RETRYG, respectively, by site-directed mutagenesis. To generate replication-incompetent viruses, plasmids for the synthesis of the HA and NA genes of GD/3, Anhui/1, K173, or VN1203, pPolIPB2(120)EGFP(336), and plasmids for the synthesis of the remaining five viral genes of PR8 were cotransfected into 293T cells together with eukaryotic protein expression plasmids for PB2, PB1, PA, and NP derived from PR8. The replication-incompetent viruses were amplified in MDCK cells that stably express the PB2 protein. Deep Sequencing Full genome sequencing of the virus stock of GD/3 was performed using next-generation sequencing. Viral RNA was extracted from the virus stock by using the QIAamp Viral RNA Mini kit (Qiagen). The complete genomes were amplified using the multi-segment RT-PCR method with the One-Step SuperScript III RT-PCR kit (Thermo Fisher Scientific) and appropriate primers (MBTuni-12 and MBTuni-13), as previously described (Zhou et al., 2009). The PCR product was purified with Agencourt AMPure XP beads (Beckman Coulter) and fragmented by NEBNext dsDNA Fragmentase (New England BioLabs) according to the manufacturer’s protocols. Then, the cDNA libraries for next-generation sequencing were prepared by using a NEBNext Ultra II DNA Library Prep kit for Illumina (New England BioLabs) and were sequenced by using a MiSeq Reagent Kit v3 150-cycle (Illumina) to perform 75-bp paired-end reads e4 Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

on a MiSeq platform. Sequence reads were mapped to the reference sequence (Global Initiative on Sharing All Influenza Data: GISAID, Isolate ID. EPI_ISL_249309) using CLC Genomics Workbench version 7.5.5 (CLC Genomics; Qiagen). The list of primers used is shown in Table S11. DNA Sequencing Viral RNA was extracted using TRIzol LS Reagent (Thermo Fisher Scientific) and reverse-transcribed to cDNA using Superscript III reverse transcriptase (Thermo Fisher Scientific) and Uni12 primer. PCR was performed with a set of primers specific for each gene segment of influenza A virus. The PCR products were purified and subjected to direct sequencing. Cycle sequencing was performed using BigDye Terminator version 3.1 Cycle Sequencing Kits (Thermo Fisher Scientific), and analyzed on an ABI Prism 3130xl Genetic Analyzer (Thermo Fisher Scientific). We estimated the level of mutation frequencies based on the height of the waves at each position on the sequencing chromatogram. The detection limit for a minor population is 10%–20%. The list of primers used is provided in Table S11. Growth Kinetics of Virus in Cell Culture Cultures of differentiated NHBE cells grown on semipermeable membrane supports were washed extensively with DMEM to remove accumulated mucus and infected in triplicate with virus at a multiplicity of infection (MOI) of 0.001 from the apical surface. The inoculum was removed after 60 min of incubation at 33
C or 37
C, and cells were further incubated at 33
C or 37
C. Samples were collected at 2, 8, 24, 48, 72, and 96 h post-infection from the apical surface. Apical collection was performed by adding 500 ml of medium to the apical surface, followed by incubation for 60 min at 33
C or 37
C, and removal of the medium from the apical surface. The titers of viruses released into the cell culture supernatant were determined by use of plaque assay in MDCK cells. Hemagglutination Inhibition (HI) Assay Ferret sera were treated with receptor-destroying enzyme (RDE II; Denka Seiken) at 37
C for 20 h, followed by RDE inactivation at 56
C for 30–60 min. The treated sera were serially diluted 2-fold with PBS in 96-well U-bottom microtiter plates and mixed with the amount of virus equivalent to eight hemagglutination units, followed by incubation at room temperature (25
C) for 60 min. After addition of 50 ml of 0.5% chicken red blood cells or turkey red blood cells, the mixtures were gently mixed and incubated at 4
C for a further 60 min. HI titers are expressed as the inverse of the highest antibody dilution that inhibited hemagglutination. Animal Experiments The sample sizes (n = 3) for the mouse and ferret studies were chosen because they have previously been shown to be sufficient to evaluate a significant difference among groups (Belser et al., 2013; Itoh et al., 2009; Liu et al., 2005; Makarova et al., 2003; Munster et al., 2009; Watanabe et al., 2013; Zhang et al., 2013; Zhu et al., 2013). For the nonhuman primate experiment, six animals were used. No method of randomization was used to determine how animals were allocated to the experimental groups and processed in this study. The investigator was not blinded to the group allocation during the experiments or when assessing the outcome. Experimental Infection of Mice Six-week-old female BALB/c mice were used in this study. Baseline body weights were measured before infection. Under isoflurane anesthesia, four mice per group were intranasally inoculated with 100, 101, 102, 103, 104, 105, or 106 PFU (50 ml) of GD/3, rGD/3NA294R, rGD/3-NA294K, or Anhui/1. Body weight and survival were monitored daily for 14 days. For virological examinations, six mice per group were intranasally infected with 103 PFU (50 ml) of the viruses and three mice per group were euthanized at 3 and 6 days post-infection. The virus titers in various organs were determined by means of plaque assays in MDCK cells. Experimental Infection of Ferrets Six- to eight-month-old female ferrets, which were serologically negative by hemagglutination inhibition assay for currently circulating human influenza viruses, were used in this study. Six ferrets per group were anesthetized intramuscularly with ketamine and xylazine (5–30 mg and 0.2–6 mg/kg of body weight, respectively), and inoculated intranasally with 106 PFU (0.5 ml) of GD/3, rGD/3-NA294R, rGD/3-NA294K, or Anhui1. Three ferrets per group were euthanized at 3 and 6 days post-infection for virological and pathological examinations. The virus titers in various organs were determined by means of plaque assays in MDCK cells. Ferret Transmission Study Pairs of ferrets were individually housed in adjacent wireframe cages that prevented direct and indirect contact between animals but allowed spread of influenza virus by respiratory droplets. Four or two per group were anesthetized intramuscularly with ketamine and xylazine (5–30 mg and 0.2–6 mg/kg of body weight, respectively), and inoculated intranasally with 106 PFU (0.5 ml) of GD/3, rGD/3NA294R, rGD/3-NA294K, Anhui/1, or CA04 (inoculated ferrets). One day after infection, four or two naive ferrets (exposed ferrets) were each placed in a cage adjacent to an infected ferret (in these cages, infected and exposed ferrets are separated by 5 cm). Nasal washes were collected from infected ferrets on day 1 after inoculation and from exposed ferrets on day 1 after co-housing, and then every other day (for up to 15 days) for virological examinations. The virus titers in nasal washes were determined by means of plaque assays in MDCK cells.

Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017 e5

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Experimental Infection of Cynomolgus Macaques Approximately 3-year-old male cynomolgus macaques (Macaca fascicularis) from Cambodia (obtained from Shin Nippon Biomedical Laboratories), weighing 2.0–3.5 kg and serologically negative by neutralization against A/Osaka/1365/2009 (H1N1pdm09), A/Kawasaki/UTK-4/2009 (seasonal H1N1), A/Kawasaki/UTK-20/2008 (H3N2), B/Tokyo/UT-E2/2008 (type B), and A/duck/Hong Kong/301/78 (H7N2) viruses, were used in this study. Six macaques were anesthetized with ketamine (5 mg/kg) and medetomidine (0.08 mg/kg) intramuscularly, and inoculated with GD/3 (107 PFU/ml) through a combination of the intratracheal (4.5 ml), intranasal (0.5 ml per nostril), ocular (0.1 ml per eye) and oral (1 ml) routes (resulting in a total infectious dose of 6.7 3 107 PFU). Body temperature was monitored at 0, 1, 3, 5, and 6 days post-infection by use of a rectal thermometer. Nasal, tracheal, and rectal swabs were collected at 1, 3, 5, and 6 days post-infection for virological examinations. Three per group of GD/3-infected macaques were euthanized at 3 and 6 days post-infection for virological and pathological examinations. Virus titers were determined by means of plaque assays in MDCK cells. Pathological Examination Excised tissues of animal organs preserved in 10% phosphate-buffered formalin were processed for paraffin embedding and cut into 3-mm-thick sections. One section from each tissue sample was stained using a standard hematoxylin and eosin procedure; another was processed for immunohistological staining with a rabbit polyclonal antibody for type A influenza nucleoprotein antigen (prepared in our laboratory) that reacts comparably with all of the viruses tested in this study (Iwasaki et al., 1999). Specific antigen-antibody reactions were visualized by means of 3,30 -diaminobenzidine tetrahydrochloride staining using the Dako Envision system (Dako Cytomation). NA Inhibition Assay In vitro NA activity of viruses was determined by using the commercially available NA-Fluor Influenza Neuraminidase Assay Kit (Thermo Fisher Scientific). In brief, diluted viruses were mixed with the indicated amounts of oseltamivir carboxylate, zanamivir, laninamivir, or peramivir and incubated at 37
C for 30 min. Methylumbelliferyl-N-acetylneuraminic acid (MUNANA) was then added as a fluorescent substrate, and the mixture was incubated at 37
C for 1 h. The reaction was stopped by adding 0.12 M Na2CO3 in 40% ethanol. The fluorescence of the solution was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm, and the IC50 values were calculated. Plaque Reduction Assay Confluent MDCK cells in 6-well plates were infected with a dilution of virus that resulted in 30–80 virus plaques per well. After a 1-h incubation, the viral inoculum was removed and the cells were overlaid with 1% agarose-containing MEM containing 0.3% bovine serum albumin in the presence of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin and different concentrations of favipiravir. The plates were incubated for 2–3 days; then, the agar overlay was removed and the cells were fixed and stained with 20% methanol and crystal violet. Visualized plaques were counted, and IC50 values were calculated by using Graphpad Prism (GraphPad Software, La Jolla, CA). Antiviral Sensitivity of Viruses in Mice Under isoflurane anesthesia, six mice per group were intranasally inoculated with 103 PFU (50 ml) of GD/3, rGD/3-NA294R, rGD/3NA294K, or Anhui/1. At 2 h after inoculation, mice were treated with the following antiviral compounds: (1) oseltamivir phosphate: 40 mg per kg per 200 ml, administered orally twice a day for 5 days; (2) zanamivir: 8 mg per kg per 50 ml, administered intranasally once daily for 5 days; (3) laninamivir: 0.75 mg per kg per 50 ml, administered intranasally once during the entire experimental course; (4) favipiravir: 60 or 150 mg per kg per 200 ml, administered orally twice a day for 5 days; or (5) saline (50 ml) as a control for intranasal treatment or methylcellulose as a control for oral treatment. For virological examinations, three mice per group were euthanized at 3 and 6 days post-infection. The virus titers in the lungs were determined by means of plaque assays in MDCK cells. Glycan Arrays Glycan array analysis was performed on a glass slide microarray containing six replicates of 129 diverse sialic acid-containing glycans, including terminal sequences and intact N-linked and O-linked glycans found on mammalian and avian glycoproteins and glycolipids (Peng et al., 2017). Viruses were amplified in MDCK cells. Supernatants collected from infected cells were centrifuged at 1,462 3 g for 30 min to remove cell debris. Viruses were inactivated by mixing the supernatants with 0.1% b-propiolactone (final concentration). Virus supernatant was laid over a cushion of 30% sucrose in PBS, ultracentrifuged at 76,755 3 g for 2 h at 4
C, and then resuspended in PBS for storage at 80
C. Virus samples were applied to the slide array, overlayed with mouse monoclonal anti-H7 or anti-H1 antibodies as the primary antibodies, and then detected with anti-mouse Alexa fluor-488-conjugated secondary antibodies. Slide scanning to detect virus bound to glycans was conducted as described previously (Peng et al., 2017). A complete list of the glycans on the array is presented in Table S7. Biolayer Interferometry The biolayer interferometry analyses were performed using the Octet Red 96 system (Forte´Bio, Menlo Park, CA, USA) in 96-well microplates, as described previously (Xiong et al., 2013a). Biotinylated glycans [Neu5Ac(a2-3)Gal(b1-4)GlcNAc (3SLN), Neu5Ac(a2-6) e6 Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

Gal(b1-4)GlcNAc (6SLN), Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc (6SLNLN), and Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3) Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc (6SLNLNLN)] coupled to polyglutamic acid-biotin were purchased from Tokyo Chemical Industry. The supernatant of replication-incompetent viruses was laid over a cushion of 30% sucrose in PBS, ultracentrifuged at 133,900 3 g for 90 min at 4
C, and then resuspended in PBS for storage at 80
C. Streptavidin biosensors (ForteBio) were immobilized with the sialoglycopolymers at 1 mg/mL in 150 mM NaCl, 10 mM HEPES (pH 7.4), 3 mM EDTA, 0.005% surfactant P20 (HBSEP), and reacted with virus samples (128 HA units) in HBS-EP containing 10 mM oseltamavir carboxylate. Association was measured for 4,000 seconds at 30
C. The data collected were processed and analyzed by using the Octet Data Analysis Software. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses were performed for organ titers of mice and ferrets by using analysis of variance (ANOVA), followed by Tukey’s test in GraphPad Prism 7; p values of < 0.05 were considered significant. We also used the R statistical package version 3.4, and the Multcomp package (Hothorn et al., 2008). We compared virus titers in mouse lung, obtained after each treatment, with the control by using one-way ANOVA, followed by Dunnett’s test. Each day (day 3, day 6) was analyzed separately, as were the different virus strains. Differences between groups were considered significant for p values < 0.05. Biosafety Statement All recombinant DNA protocols were approved by the University of Wisconsin-Madison’s Institutional Biosafety Committee after risk assessments were conducted by the Office of Biological Safety, and by the University of Tokyo’s Subcommittee on Living Modified Organisms, and, when required, by the competent minister of Japan. All experiments were approved by the respective committees at the University of Tokyo and by the University of Wisconsin-Madison’s Institutional Biosafety Committee (IBC). When virus transmission was detected, the University of Wisconsin’s Alternate Responsible Official (ARO) and Institutional Contact for Dual Use Research (ICDUR) was contacted and a risk assessment was performed. All practices and procedures used for additional experiments followed the requirements of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules for working with mammalian-transmissible H7N9 viruses. The ARO/ ICDUR was kept informed of the research results. This manuscript was reviewed by the University of Wisconsin-Madison Dual Use Research of Concern (DURC) Subcommittee in accordance with the United States Government September 2014 DURC Policy, which concluded that the studies described herein do not constitute DURC since the natural virus isolates were not modified or sequentially passed in our laboratory. In addition, the University of Wisconsin-Madison Biosecurity Task Force regularly reviews the research, policies, and practices of research conducted with pathogens of high consequence at the institution. This task force has a diverse skill set and provides support in the areas of biosafety, facilities, compliance, security, law, and health. Members of the Biosecurity Task Force are in frequent contact with the principal investigator and laboratory personnel to provide oversight and assure biosecurity. All experiments with H7N9 viruses were performed in enhanced biosafety level 3 laboratories at the University of Tokyo (Tokyo, Japan), which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan, or in biosafety level 3 agricultural (BSL-3Ag) laboratories at the University of Wisconsin-Madison approved for such use by the Centers for Disease Control and Prevention (CDC) and Animal and Plant Health Inspection Service (APHIS). Ferret transmission and mouse virulence studies were conducted in enhanced BSL-3 containment at the University of Tokyo by PhD-level scientists who are highly experienced in such studies. In vitro experiments were conducted in Class II biological safety cabinets and transmission experiments were conducted in HEPA-filtered ferret isolators. Staff working in enhanced BSL-3 and BSL-3Ag wear disposable overalls and powered air-purifying respirators. The enhanced BSL-3 facility at the University of Tokyo includes controlled access, exit through a shower change room, effluent decontamination, negative air-pressure, double-door autoclaves, HEPA-filtered supply and exhaust air, and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets. The structure is pressure-decay tested regularly. All personnel complete biosafety and BSL-3 training before participating in BSL-3-level experiments. Refresher training is scheduled on a regular basis. Select Agent virus inventory, secured behind two physical barriers, is checked regularly. Virus inventory is submitted once a year to the Ministry of Agriculture, Forestry and Fisheries, Japan The BSL-3Ag facility at University of Wisconsin-Madison was designed to exceed the standards outlined in Biosafety in Microbiological and Biomedical Laboratories (5th edition; http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf). Features include controlled access, entry/exit through a shower change room, effluent decontamination, negative air-pressure, double-door autoclaves, gas decontamination ports, HEPA-filtered supply and double-HEPA-filtered exhaust air, double-gasketed watertight and airtight seals, and airtight dampers on all ductwork. The structure is pressure-decay tested regularly. The University of WisconsinMadison facility has a dedicated alarm system that monitors all building controls (500 possible alerts). Redundancies and emergency resources are built into the facility, including two air handlers, two compressors, two filters wherever filters are needed, two effluent sterilization tanks, two power feeds to the building, an emergency generator in case of a power failure, and other physical containment measures in the facility that operate without power. Biosecurity monitoring of the facility is ongoing. All personnel undergo Select Agent security risk assessment by the United States Criminal Justice Information Services Division and complete rigorous biosafety, BSL-3, and Select Agent training before participating in BSL-3-level experiments. Refresher training, including drills and review of emergency plans, is scheduled on a regular basis. The principal investigator participates in training sessions and emphasizes compliance to maintain safe operations and a responsible research environment. The laboratory occupational Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017 e7

Please cite this article in press as: Imai et al., A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets, Cell Host & Microbe (2017), https://doi.org/10.1016/j.chom.2017.09.008

health plan is in compliance with the University of Wisconsin-Madison Occupational Health Program. Select Agent virus inventory, secured behind two physical barriers, is checked monthly and documentation is submitted to the University of Wisconsin-Madison Select Agent Program Manager. Virus inventory is submitted 1–2 times per year to the file holder in the Division of Select Agents and Toxins at the CDC. The research program, procedures, occupational health plan, documentation, security, and facilities are reviewed annually by the University of Wisconsin-Madison Responsible Official and at regular intervals by the CDC and the APHIS as part of the University of Wisconsin-Madison Select Agent Program

e8 Cell Host & Microbe 22, 1–12.e1–e8, November 8, 2017