Appendix C

The Two Published H5N1 Papers

“Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets,” Masaki Imai, et al., Nature 420 (486). Copyright 2012. Mcmillan Publishers Limited. All rights reserved.

 

“Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets,” Sander Herfst, et al., Science 336 (June 22, 2012):1543. Reprinted with permission from AAAS.



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Appendix C The Two Published H5N1 Papers “Experimental adaptation of an influenza H5 HA confers respira- tory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets,” Masaki Imai, et al., Nature 420 (486). Copyright 2012. Mcmillan Publish- ers Limited. All rights reserved. “Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets,” Sander Herfst, et al., Science 336 (June 22, 2012):1543. Reprinted with permission from AAAS. 81

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82 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA LETTER doi:10.1038/nature10831 Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets Masaki Imai1, Tokiko Watanabe1,2, Masato Hatta1, Subash C. Das1, Makoto Ozawa1,3, Kyoko Shinya4, Gongxun Zhong1, Anthony Hanson1, Hiroaki Katsura5, Shinji Watanabe1,2, Chengjun Li1, Eiryo Kawakami2, Shinya Yamada5, Maki Kiso5, Yasuo Suzuki6, Eileen A. Maher1, Gabriele Neumann1 & Yoshihiro Kawaoka1,2,3,5 Highly pathogenic avian H5N1 influenza A viruses occasionally before a pandemic. Therefore, we studied the molecular features that infect humans, but currently do not transmit efficiently among would render H5-HA-possessing viruses transmissible in mammals. humans. The viral haemagglutinin (HA) protein is a known Previous studies suggested that HA has a major role in host-range host-range determinant as it mediates virus binding to host- restriction of influenza A viruses1–3. The HA of human isolates preferen- specific cellular receptors1–3. Here we assess the molecular changes tially recognizes sialic acid linked to galactose by a2,6-linkages in HA that would allow a virus possessing subtype H5 HA to be (Siaa2,6Gal), whereas the HA of avian isolates preferentially recognizes transmissible among mammals. We identified a reassortant H5 sialic acid linked to galactose by a2,3-linkages (Siaa2,3Gal)3. A small HA/H1N1 virus—comprising H5 HA (from an H5N1 virus) with number of avian H5N1 viruses isolated from humans show limited four mutations and the remaining seven gene segments from a binding to human-type receptors, a property conferred by several amino 2009 pandemic H1N1 virus—that was capable of droplet transmis- acid changes in HA4–9. None of the H5N1 viruses tested transmitted sion in a ferret model. The transmissible H5 reassortant virus efficiently in a ferret model10–13, although, while our paper was under preferentially recognized human-type receptors, replicated effi- review, one study14 reported that a virus with a mutant H5 HA and a ciently in ferrets, caused lung lesions and weight loss, but was neuraminidase (NA) of a human virus in the H5N1 virus background not highly pathogenic and did not cause mortality. These results caused respiratory droplet transmission in one of two contact ferrets. indicate that H5 HA can convert to an HA that supports efficient To identify novel mutations in avian H5 HAs that confer human- viral transmission in mammals; however, we do not know whether type receptor-binding preference, we introduced random mutations the four mutations in the H5 HA identified here would render a into the globular head (amino acids 120–259 (H3 numbering), which wholly avian H5N1 virus transmissible. The genetic origin of the includes the receptor-binding pocket) of A/Vietnam/1203/2004 remaining seven viral gene segments may also critically contribute (H5N1; VN1203) HA (Supplementary Fig. 1). Although this virus to transmissibility in mammals. Nevertheless, as H5N1 viruses was isolated from a human, its HA retains avian-type receptor-binding continue to evolve and infect humans, receptor-binding variants properties6,15. We also replaced the multibasic HA cleavage sequence of H5N1 viruses with pandemic potential, including avian–human with a non-virulent-type cleavage sequence, allowing us to per- reassortant viruses as tested here, may emerge. Our findings form studies in biosafety level 2 containment (http://www.who.int/ emphasize the need to prepare for potential pandemics caused by csr/resources/publications/influenza/influenzaRMD2003_5.pdf). The influenza viruses possessing H5 HA, and will help individuals con- mutated polymerase chain reaction (PCR) products were cloned into ducting surveillance in regions with circulating H5N1 viruses to RNA polymerase I plasmids16 containing the VN1203 HA comple- recognize key residues that predict the pandemic potential of iso- mentary DNA, which resulted in Escherichia coli libraries representing lates, which will inform the development, production and distri- the randomly generated HA variants. Sequence analysis of 48 bution of effective countermeasures. randomly selected clones indicated an average of 1.0 amino acid Although H5N1 viruses continue to cause outbreaks in poultry and changes per HA globular head (data not shown). To generate an there are cases of human infection in Indonesia, Vietnam, Egypt and H5N1 virus library, plasmids for the synthesis of the mutated HA gene elsewhere (http://www.who.int/influenza/human_animal_interface/H5N1_ and the unmodified NA gene of VN1203 were transfected into human cumulative_table_archives/en/index.html), they have not acquired the embryonic kidney (293T) cells together with plasmids for the synthesis ability to cause human-to-human transmission. Investment in H5N1 of the six remaining viral genes of A/Puerto Rico/8/34 (H1N1; PR8), a vaccines has therefore been questioned. However, because humans laboratory-adapted human influenza A virus. lack immunity to influenza viruses possessing an H5 HA, the emergence Turkey red blood cells (TRBCs; which possess both Siaa2,6Gal and of a transmissible H5-HA-possessing virus would probably cause a Siaa2,3Gal on their surface (data not shown)) were treated with pandemic. To prepare better for such a scenario, it is critical that we Salmonella enterica serovar Typhimurium LT2 sialidase, which pref- understand the molecular changes that may render H5-HA-possessing erentially removes a2,3-linked sialic acid (that is, avian-type receptors), viruses transmissible in mammals. Such knowledge would allow us to creating TRBCs that predominantly possess Siaa2,6Gal on the cell monitor circulating or newly emerging variants for their pandemic surface (Siaa2,6-TRBCs; Supplementary Fig. 2). The virus library potential, focus eradication efforts on viruses that already have was then adsorbed to Siaa2,6-TRBCs at 4 uC and extensively washed acquired subsets of molecular changes critical for transmission in to remove nonspecifically or weakly bound viruses. Bound viruses were mammals, stockpile antiviral compounds in regions where such viruses eluted by incubation at 37 uC for 30 min, and then diluted to approxi- circulate, and initiate vaccine generation and large-scale production mately ,0.5 viruses per well (on the basis of a pilot experiment that 1 Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. 2ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan. 3Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 4Department of Microbiology and Infectious Diseases, Kobe University, Hyogo 650-0017, Japan. 5Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 6Health Science Hills, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan. 4 2 0 | N AT U R E | VO L 4 8 6 | 2 1 J U N E 2 0 1 2 ©2012 Macmillan Publishers Limited. All rights reserved

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APPENDIX C 83 LETTER RESEARCH assessed the approximate number of eluted viruses). We screened one- human-type receptors. For the recreated variants, haemagglutination third of the library (that is, 2.1 3 106 viruses) in three separate selection titres were higher and slightly different from the initial characteriza- experiments (that is, 0.7 3 106 viruses per experiment) and isolated tion, which we attribute to biological differences (the initial character- 370 viruses that bound to Siaa2,6-TRBCs (Supplementary Fig. 2). ization was carried out with non-concentrated cell culture supernatant Individual viruses were then grown in Madin-Darby canine kidney and potentially mixed virus populations, whereas the recreated viruses (MDCK) cells modified to overexpress Siaa2,6Gal (AX4 cells17), and were concentrated and purified) and to experimental differences (that screened again for their ability to agglutinate Siaa2,6-TRBCs (Sup- is, differences between the TRBC batches or the efficiency of a2,3- plementary Fig. 2). The parental control virus (designated VN1203/ sialidase treatment, or both). Collectively, however, these experiments PR8) with avian-type receptor-binding specificity agglutinated untreated demonstrate that this random mutagenesis approach allows the TRBCs (which possess both human- and avian-type receptors on their identification of hitherto unrecognized amino acid substitutions that surface), but not TRBCs possessing predominantly human-type recep- permit avian virus HAs to bind to human-type receptors. tors (Siaa2,6-TRBCs; Supplementary Table 1). By contrast, of the 370 To characterize further the receptor-binding properties of the viruses originally isolated, nine agglutinated Siaa2,6-TRBCs, albeit selected variants, we used solid-phase binding assays in which with different efficiencies (Supplementary Table 1). All nine viruses sialylglycopolymers were absorbed to plates, which were then possessed mutations in the region targeted for random mutagenesis; incubated with virus (Fig. 2a). A virus possessing the HA and NA one mutant also possessed an additional mutation (E119G) in an area genes of the seasonal human A/Kawasaki/173/2001 (H1N1; K173) that was not targeted for mutation. Most of the mutations clustered virus and the remaining genes from PR8 (K173/PR8) served as a around the receptor-binding pocket (Fig. 1a). Several of the selected control virus with typical human-type receptor specificity. Indeed, viruses possessed mutations known to increase binding to human- K173/PR8 preferentially bound to Siaa2,6Gal. In contrast, VN1203/ type receptors, including N186K (ref. 9), S227N (ref. 5) and Q226L PR8 bound to only Siaa2,3Gal. As reported elsewhere6,15, the Q226L/ (which confers human-type receptor binding together with G228S)15 G228S mutations led to increased binding to Siaa2,6Gal. Variants (all shown in blue in Fig. 1a). The identification of known deter- I202T/R220S, W153R/T160I, N169I/H184L/I217M and H130Q/ minants of human-type receptor-binding specificity from a library of K157E resembled VN1203/PR8 in their binding to glycans, despite random mutants validates our approach. Notably, our screen also the fact that these mutants weakly agglutinated Siaa2,6-TRBCs (see identified mutations not previously associated with receptor-binding Supplementary Table 1). These viruses may have bound to glycans on specificity. TRBCs that were different from Siaa2,6Galb1,4GlcNAc used in this Although viruses were diluted to ,0.5 viruses per well for amplifica- study. However, variants N186K/M230I, S227N/G228A and Q226L/ tion in AX4 cells, we cannot exclude the possibility that some wells were E231G showed an appreciable increase in binding to Siaa2,6Gal but infected with more than one virus, resulting in mixed populations. also retained binding capacity for Siaa2,3Gal. Of all of the variants To confirm the significance of the identified mutations in HA for tested, only E119G/V152I/N224K/Q226L exhibited specificity for only human-type receptor binding, the mutations were engineered into a Siaa2,6Gal. Thus, only one H5 HA variant with receptor-binding VN1203/PR8 virus (possessing an avirulent HA cleavage site sequence, capability akin to that of seasonal influenza viruses was isolated from as described earlier). All nine mutants were generated; however, after the library screen of 2.1 3 106 viruses. To identify the amino acid two passages in MDCK cells, the S136N mutation reverted to the wild- change(s) responsible for the conversion from Siaa2,3Gal to type sequence. This mutant was excluded from further evaluation. Siaa2,6Gal recognition in the E119G/V152I/N224K/Q226L virus First, we confirmed the binding of the remaining eight variants to HA, we tested the amino acid changes at positions 119, 152, 224 and Siaa2,6-TRBCs (Supplementary Table 1). For comparison, we 226 individually and in various combinations. Solid-phase binding included a VN1203/PR8 virus with two changes in its HA (Q226L assays demonstrated that the N224K/Q226L combination is critical and G228S) previously shown to have increased binding to for the shift from Siaa2,3Gal to Siaa2,6Gal recognition (Fig. 2b); Siaa2,6Gal6,15. Indeed, compared to the wild-type VN1203/PR8 virus, Q226L in combination with V152I also conferred weak binding to the Q226L/G228S mutant displayed an increased ability to bind to a2,6-glycans. a Modelled human receptor b N158D N158D K193N Modelled human receptor 190-helix Q226L N186K N224K G228A A242T S227N T318I V152I E231G Q226L M230I G225E 130-loop Fusion peptide 220-loop N224K E119G Figure 1 | Localization of amino acid changes identified in this study on the human reassortant viruses during replication and/or transmission in ferrets are three-dimensional structure of the monomer of VN1203 HA (Protein Data shown in red. b, The positions of four mutations in the HA of H5 transmissible Bank accession 2FK0)15. a, Close-up view of the globular head of VN1203 HA. reassortant mutant virus, HA(N158D/N224K/Q226L/T318I)/CA04, are Mutations known to increase affinity to human-type receptors are shown in highlighted in red. The fusion peptide of HA is shown in cyan. All mutations are blue. Amino acid changes not previously known to affect receptor binding are shown with H3 numbering. Images were created with MacPymol (http:// shown in green. Additional mutations that occurred in the HA of H5 avian– www.pymol.org/). 2 1 J U N E 2 0 1 2 | VO L 4 8 6 | N AT U R E | 4 2 1 ©2012 Macmillan Publishers Limited. All rights reserved

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84 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA RESEARCH LETTER a 1.6 K173/PR8 VN1203/PR8 Q226L/G228S c Trachea Alveoli Trachea Alveoli 1.2 0.8 K173/ N224K/ 0.4 PR8 Q226L 0 1.6 N186K/M230I S227N/G228A I202T/ 1.2 R220S 0.8 Absorbance (490 nm) 0.4 N158D/ VN1203/ 0 N224K/ 1.6 Q226L/E231G PR8 W153R/T160I E119G/V152I/ Q226L 1.2 N224K/Q226L 0.8 0.4 0 N158D/ 2.50 10.0 0.001 0.002 0.010 0.039 0.156 0.625 1.6 N186K/ N224K/ N169I/H184L/ H130Q/K157E 1.2 I217M M230I Q226L/ 0.8 T318I 0.4 0 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 2.50 10.0 0.001 0.002 0.010 0.039 0.156 0.625 S227N/ T318I Sialylglycopolymer (μg ml–1) G228A b 1.6 E119G/V152I/ E119G/V152I/ E119G/V152I/ 1.2 N224K/Q226L N224K Q226L 0.8 0.4 d Q226L/ 0 Absorbance (490 nm) 1.6 E231G N158D/N224K/ N158D/N224K/ T318I Absorbance (490 nm) 1.6 1.2 Q226L E119G/N224K/ V152I/N224K/ E119G/Q226L Q226L/T318I 1.2 Q226L Q226L 0.8 0.8 0.4 0.4 E119G/ 0 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 0 V152I/ 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 1.6 V152I/Q226L N224K/Q226L N224K/ 1.2 Q226L Sialylglycopolymer (μg ml–1) 0.8 0.4 0 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 0.001 0.002 0.010 0.039 0.156 0.625 2.50 10.0 Sialylglycopolymer (μg ml–1) Figure 2 | Characterization of the receptor-binding properties of isolated with human tissue sections and then stained with either anti-K173 antiserum viruses. a, Binding of VN1203 mutants to sialylglycopolymers in solid-phase (green) or anti-VN1203 HA antibodies (green). All sections were subsequently binding assays. A human virus (K173/PR8), an avian virus (VN1203/PR8) and incubated with labelled secondary antibodies and Hoechst dye (blue). mutant VN1203/PR8 viruses were compared for their ability to bind to d, Characterization of the receptor-binding properties of N158D/N224K/ sialylglycopolymers containing either a2,3-linked (blue) or a2,6-linked (red) Q226L, N158D/N224K/Q226L/T318I and T318I viruses. The direct binding of sialic acids. b, Identification of mutations that confer binding to human-type virus to sialylglycopolymers containing either a2,3-linked (blue) or a2,6-linked receptors. c, Binding of VN1203 mutant viruses to human respiratory tissues. (red) sialic acids was determined as described in panel a. K173/PR8, VN1203/PR8 and mutant VN1203/PR8 viruses were incubated To assess the effect of enhanced a2,6-glycan recognition on the mutants exhibited strong binding to the ciliated epithelial cells of the attachment of viruses to human respiratory tracts, sections of tracheal trachea (Fig. 2c and Supplementary Fig. 3). By contrast, the N186K/ and lung tissues were exposed to K173/PR8 (human-type receptor M230I, S227N/G228A and Q226L/E231G mutants displayed little-to- binder), VN1203/PR8 (avian-type receptor binder) and mutant no binding to tracheal epithelia (Fig. 2c), despite their binding to VN1203/PR8 viruses (Fig. 2c). Because the N186K/M230I, S227N/ Siaa2,6Gal (Fig. 2a). A number of sialylated oligosaccharides with G228A, Q226L/E231G, E119G/V152I/N224K/Q226L and N224K/ differing branching patterns and chain lengths are thought to be Q226L mutants exhibited appreciable binding to Siaa2,6Gal (Fig. 2a, b), present on the cell surface19. We therefore speculate that the mutants the attachment of these mutants was also tested. On tracheal sections, can recognize a short glycan structure such as Siaa2,6Galb1,4GlcNAc, the K173/PR8 virus bound extensively to ciliated epithelial cells (Fig. 2c but may not recognize longer, more complex glycan structures, which and Supplementary Fig. 3), whereas the VN1203/PR8 virus bound are possibly required for binding to human tracheal epithelium. On the poorly. By contrast, on lung sections, both viruses bound extensively other hand, all mutants bound to alveolar epithelial cells (both type I to the alveolar epithelial surface (both type I and II pneumocytes; Fig. 2c and II pneumocytes; Fig. 2c and Supplementary Fig. 4). When the and Supplementary Fig. 4). The binding patterns of these viruses cor- tissue sections were pre-treated with Arthrobacter ureafaciens sialidase relate with the distribution of Siaa2,3Gal (that is, avian-type receptors; (which cleaves all non-reducing terminally branched and unbranched present in lung epithelia) and Siaa2,6Gal (that is, human-type recep- sialic acids), virus binding to the tissues was substantially reduced tors; present in both trachea and lung epithelia) on the tissues, as (Supplementary Fig. 6a–c), confirming the sialic acid binding specifi- observed with lectin staining18 (Supplementary Fig. 5). Like the human city of the virus. These data indicate that alterations in the receptor K173/PR8 virus, the E119G/V152I/N224K/Q226L and N224K/Q226L specificity of the E119G/V152I/N224K/Q226L and N224K/Q226L 4 2 2 | N AT U R E | VO L 4 8 6 | 2 1 J U N E 2 0 1 2 ©2012 Macmillan Publishers Limited. All rights reserved

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APPENDIX C 85 LETTER RESEARCH mutants have profound effects on virus attachment to human respir- Ferret 1 Ferret 2 Ferret 3 Mean atory epithelium. Day 3 In an avian H3 HA, the Q226L mutation changed the binding Nasal turbinates Trachea Lung preference from avian- to human-type20. A previous study found that 10 the Q226L mutation on an H5 HA does not confer efficient binding to Virus titre log10(p.f.u. g–1) a2,6-glycans in a glycan array15; however, when tested in combination 8 with G228S, increased binding to human-type receptors, but not a complete switch from avian- to human-type receptor-binding specifi- 6 * city, was observed15. By contrast, here we found that Q226L in com- 4 * * * bination with N224K resulted in a switch from Siaa2,3Gal to * Siaa2,6Gal binding in an H5 HA and allowed virus binding to human 2 tracheal epithelia (Fig. 2c). The receptor-binding domain of HA is formed by the 190-helix at the top of HA, the 220-loop at the edge 0 of the globular head, and the 130-loop at the other edge of the globular head (Fig. 1a). Crystal structure analysis revealed that the 220-loop of Day 6 avian H5 HA is closer to the opposing 130-loop than in human H3 HA, 10 Nasal turbinates Trachea Lung indicating that a wider binding site for human H3 HA, compared to Virus titre log10(p.f.u. g–1) that of avian H5 HA, may be required to optimize contacts with the 8 larger Siaa2,6-glycans21. N224 lies on the turn leading into the 220- loop, adjacent to position 226 (Fig. 1a). Replacement of N224 may alter 6 the orientation of the 220-loop and thus optimize contacts between L226 and Siaa2,6Gal-containing receptors, thereby increasing the 4 preference for a2,6 linkages. 2 Recent studies reported that 2009 pandemic H1N1 and H5N1 viruses show high genetic compatibility22,23. These two viruses have 0 been isolated from pigs24–28, which have been considered as ‘mixing /N D/N 24K 22 /C 4 4K 26 /CA 4 /N D/N 24K 22 3/C 4 4K 24 /Q2 6L)/CA04 04 4K 24 Q22 L)/C 04 04 4K 24 /Q22 L)/C 04 04 /T3 L)/ 04 /T3 L)/ 04 /T3 L)/ A04 31 /CA 4 rgT 18I)/ A04 rgT 18I) A04 22 2 )/ 04 8I/ 04 22 Q2 L)/C 04 8I/ 04 22 2 )/C 4 8I/ 04 8D 58 2 /Q 03 A0 8D 58 N22 K/Q2 203 A0 58 (N2 4K/Q 120 CA0 rgT 18I) CA0 /Q K/Q 6L A0 CA CA A CA 6L 26 CA A /Q K/Q 26L A A 31 CA 31 /CA vessels’ for the reassortment of avian, swine and human strains. Thus, 15 1 g(N 4K 12 C C C C (N HA(N r N22 rgVN rg (N A(N rg 224 VN1 rg (N A(N rg N22 gVN rg the coexistence of H5N1 and 2009 pandemic H1N1 viruses could pro- 6 6L 26 6L 26 6 vide an opportunity for the generation of transmissible H5 avian– / /Q K/ human reassortants in mammals. Therefore, we generated reassortant I/N rg r 22 2 22 N2 22 2 viruses possessing the mutant VN1203 HAs generated above, and the ( /N D/ 2I/ I/ 52 52 seven remaining gene segments from a prototype 2009 pandemic 15 V1 V1 15 1 15 1 /V H1N1 virus (A/California/04/2009, CA04). Experiments with viruses G/ G/ 8D 9G 19 19 possessing the wild-type HA cleavage site were performed in enhanced HA H HA H 11 E1 E1 E biosafety level 3 (BSL31) containment laboratories approved for such rg( rg( rg( HA use by the Centers for Disease Control and Prevention (CDC) and the United States Department of Agriculture (USDA). Because efficient Figure 3 | Virus replication in respiratory organs. Ferrets were infected intranasally with 106 p.f.u. of virus. Three ferrets per group were killed on days 3 human-to-human transmission is a critical feature of pandemic and 6 after infection for virus titration. Virus titres in nasal turbinates, trachea influenza viruses, we examined the growth and transmissibility of and lung were determined by use of a plaque assay on MDCK cells. Horizontal reassortant viruses in ferrets, which are widely accepted as an animal bars show the mean. Asterisks indicate virus titres significantly different from model for influenza virus transmissibility and pathogenesis studies. that of rgCA04 (Dunnett’s test; P , 0.05). Because the E119G/V152I/N224K/Q226L and N224K/Q226L variants bound extensively to human tracheal epithelia (Fig. 2c), we generated V152I/N224K/Q226L)/CA04 than in animals inoculated with rgCA04 by reverse genetics (rg) three H5 reassortant viruses possessing the (Dunnett’s test; P 5 0.0002; Fig. 3). Notably, rgVN1203/CA04 (avian- VN1203 HA or mutant HAs (all with the wild-type multibasic cleavage type receptor binder) replicated efficiently in nasal turbinates of site) and the remaining genes from the CA04 virus. The VN1203 HA ferrets, which have a similar sialic acid receptor distribution pattern mutants tested included the one containing four mutations, E119G, to that of the human respiratory tract29,30. The reason for this discrep- V152I, N224K and Q226L (designated rg(E119G/V152I/N224K/ ancy is unclear; however, replication of avian H5N1 viruses in ferret Q226L)/CA04), and another containing two mutations, N224K and nasal turbinates has been reported12,13. Q226L (designated rg(N224K/Q226L)/CA04). Although virus titres in respiratory organs were generally lower on To determine whether the introduced HA mutations affected the day 6 after infection than on day 3 after infection, rg(N224K/Q226L)/ replication of the H5 reassortant viruses, six ferrets were inoculated CA04 still showed high levels of replication at day 6 after infection; intranasally with 106 plaque-forming units (p.f.u.) of virus. On day 3 titres in nasal turbinates ranged from 104.6 to 108.1 p.f.u. g21 (Fig. 3). after infection, a recombinant virus whose genes all came from CA04, Sequence analysis of viruses in nasal turbinates on day 6 after infection rgCA04, replicated efficiently in the respiratory organs of infected revealed that viruses in ferret 2 and ferret 3 possessed N158D and animals, and was isolated from the colon, but not from any other N158K mutations in their HA (in addition to the original two muta- organs tested (Fig. 3 and Supplementary Table 2). A virus possessing tions), respectively, leading to the loss of the glycosylation site at posi- H5 VN1203 HA and the remaining genes from CA04 (designated tion 158 (that is, 158N-S-T to 158D-S-T or 158K-S-T; Fig. 1a and rgVN1203/CA04) replicated to titres comparable to those of rgCA04 Supplementary Table 3). In nasal turbinates on day 6 after infection, in nasal turbinates, but substantially less in the lungs. By contrast, the the titre of the virus with the N158D/N224K/Q226L mutations two H5 reassortant viruses with HA mutations (rg(E119G/V152I/ (108.1 p.f.u. g21; see Fig. 3, ferret 2 of rg(N224K/Q226L)/CA04) was N224K/Q226L)/CA04 and rg(N224K/Q226L)/CA04) were severely approximately four orders of magnitude higher than that of the limited in their replicative ability in trachea. Although virus titres in original rg(N224K/Q226L)/CA04 (104.6 p.f.u. g21; Fig. 3, ferret 1 of nasal turbinates and lung were not statistically different between rg(N224K/Q226L)/CA04), whereas the virus with the N158K/ rg(N224K/Q226L)/CA04 and rgCA04, the virus titre in nasal turbi- N224K/Q226L mutations (105.6 p.f.u. g21; Fig. 3, ferret 3 of nates was significantly lower in animals inoculated with rg(E119G/ rg(N224K/Q226L)/CA04) grew to one order of magnitude higher than 2 1 J U N E 2 0 1 2 | VO L 4 8 6 | N AT U R E | 4 2 3 ©2012 Macmillan Publishers Limited. All rights reserved

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86 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA RESEARCH LETTER the original mutant. These data indicate that the additional mutation (K193N and A242S) (Fig. 1a), whereas those derived from the contact N158D improved the replication of rg(N224K/Q226L)/CA04 in ferrets. animals of pair 2 contained a single change in HA (T318I) (Fig. 1b), To test the effect of this mutation on the replication of H5 reassortant indicating that additional changes in HA occurred during the infection viruses in ferrets, we examined the replicative ability of a virus with of ferrets with HA(N158D/N224K/Q226L)/CA04. No mutations in the the triple N158D/N224K/Q226L HA substitutions in ferrets. This remaining genes were detected in any of these viruses from nasal washes HA(N158D/N224K/Q226L)/CA04 virus replicated efficiently in compared with the CA04 virus sequences. infected animals, except in the trachea (Fig. 3 and Supplementary Because HA(N158D/N224K/Q226L)/CA04 was isolated from only Table 2). On day 3 after infection, this virus was isolated from the brain one-third of the contact animals, we isolated a virus from the nasal of two of the three animals tested, although we did not observe neuro- wash of the contact ferret that shed a high titre (107.5 p.f.u. ml21) of logical signs in these animals. These results indicate that the N158D virus on day 7 after contact (pair 2) (Fig. 4d) to evaluate the replication mutation contributed to the efficient growth in the nasal turbinates of and transmissibility of that virus in ferrets. This mutant virus, desig- ferrets of an H5 reassortant virus with the N224K/Q226L mutations. nated HA(N158D/N224K/Q226L/T318I)/CA04, replicated efficiently Removal of the glycosylation site at position 158 has been reported to in the nasal turbinates and was isolated from brain tissue (Fig. 3 and result in enhanced binding of H5N1 viruses to human-type receptors Supplementary Table 2). In the transmission study, four of the six in combination with the Q226L/G228S mutations7. A previous study contact ferrets were positive for virus between days 3 and 7 after showed that H5N1 viruses lacking this glycosylation site transmit effi- contact, and all contact animals were seropositive; no animals died ciently by direct contact among guinea-pigs31. By contrast, H5N1 in the course of the transmission experiments (Table 1; Fig. 4e and viruses that acquire this glycosylation site lose the ability to transmit Supplementary Fig. 8). Notably, this transmission pattern is compar- among guinea-pigs. Therefore, we speculated that the loss of the gly- able to that of the 1918 pandemic H1N1 virus when tested under the cosylation site in HA(N158D/N224K/Q226L)/CA04 virus may affect same experimental conditions; the 1918 pandemic virus was recovered its transmissibility in ferrets. from the nasal wash of two of three contact animals (our own unpub- To assess the ability of H5 reassortant viruses with human-type lished data). Sequence comparison of viruses from inoculated and receptor specificity to transmit between ferrets, we placed naive ferrets contact animals identified mutations at positions 225 and 242 as well in wireframe cages next to ferrets inoculated with 106 p.f.u. of rgCA04, as a reversion at position 224 (Fig. 1a and Supplementary Table 5) (in rgVN1203/CA04, rg(N224K/Q226L)/CA04, or HA(N158D/N224K/ addition to the original four mutations) although the 224 reversion was Q226L)/CA04 (Supplementary Fig. 7). Similar to previous experi- found only in viruses from inoculated ferrets. Collectively, these find- ments32, rgCA04 was efficiently transmitted via respiratory droplets ings demonstrate that four amino acid substitutions (N158D/N224K/ to all three contact ferrets, as evidenced by the detection of virus in Q226L/T318I) in H5 HA confer efficient respiratory droplet transmis- nasal washes and haemagglutination inhibition (HI) antibody in these sion in ferrets to a virus possessing an H5 HA in a 2009 pandemic animals (Table 1 and Fig. 4). By contrast, rgVN1203/CA04 and H1N1 backbone. We also confirmed that recombinant viruses posses- rg(N224K/Q226L)/CA04 were not transmitted; neither virus shedding sing the three HA mutations N158D, N224K and Q226L, or the four nor seroconversion was detected in any contact animals, despite the HA mutations N158D, N224K, Q226L and T318I, and the NA of binding of the latter to Siaa2,6Gal. This result was consistent with that VN1203 in a PR8 backgrand (designated N158D/N224K/Q226L or of previous studies in which human-type receptor recognition was N158D/N224K/Q226L/T318I, respectively) preferentially bind to shown to be necessary but not sufficient for respiratory droplet trans- Siaa2,6Gal and attach to human tracheal epithelia (Fig. 2c, d). mission of an H5N1 virus in a ferret model12,14. In the HA(N158D/ HA(N158D/N224K/Q226L/T318I)/CA04 transmitted by respir- N224K/Q226L)/CA04-inoculated group, virus was recovered from atory droplet more efficiently than HA(N158D/N224K/Q226L)/ two of the six contact ferrets (pairs 1 and 2) between days 5 and 7 after CA04, raising the possibility that the T318I mutation is involved in contact. Moreover, seroconversion was detected in five animals the efficient transmission of avian H5N1/pandemic H1N1 reassor- including those from which virus was recovered. No animals died in tants. To explore the functional role of this mutation in respiratory the course of these transmission experiments. This finding demon- droplet transmission, we generated an H5 reassortant expressing the strates the generation of an H5 HA that supports virus transmission by H5 HA with the T318I mutation and examined its receptor-binding respiratory droplets among ferrets. specificity and transmissibility. This reassortant (designated rgT318I/ To determine whether additional mutations occurred in the HA of CA04) bound to only Siaa2,3Gal and showed little binding to human HA(N158D/N224K/Q226L)/CA04 during transmission, viral RNA was tracheal epithelia (Fig. 2c, d). rgT318I/CA04 did not transmit via analysed from nasal washes of inoculated and contact ferrets (Fig. 4 and respiratory droplet among ferrets (Table 1 and Fig. 4f), although it Supplementary Table 4). On day 5 after infection, the A242S and T318I replicated in nasal turbinates and trachea as efficiently as rgCA04 mutations in HA were present in five (pairs 1, 3, 4, 5 and 6) and one (pair (Fig. 3 and Supplementary Table 2). These results indicate that the 2) of the six inoculated animals, respectively. Viruses derived from the T318I mutation alone is not sufficient for H5 reassortant viruses to contact animals of pair 1 on day 7 after contact had two changes in HA transmit efficiently among ferrets. Table 1 | Transmission in ferrets inoculated with H5 avian–human reassortant viruses Virus Inoculated ferrets Contact ferrets Weight loss Peak virus titre in nasal wash Seroconversion Virus detection in Seroconversion (%)* (mean log10(p.f.u. ml21)) (positive and total numbers) nasal wash (positive (positive and total numbers) (days after inoculation) (HI titre){ and total numbers) (HI titre) rgCA04 3 of 3 (15.1) 7.5 (1) 3 of 3 ($1,280, $1,280, $1,280) 3 of 3 3 of 3 ($1,280, $1,280, $1,280) rgVN1203/CA04 3 of 3 (5.9) 5.3 (5) 3 of 3 (80, 40, 80) 0 of 3 0 of 3 (,10, ,10, ,10) rg(N224K/Q226L)/CA04 2 of 3 (7.8){ 3.9 (5) 3 of 3 ($1,280, $1,280, $1,280) 0 of 3 0 of 3 (,10, ,10, ,10) HA(N158D/N224K/Q226L)/ 6 of 6 (5.7) 6.7 (3) 6 of 6 (640, $1,280, $1,280, 640, 2 of 6 5 of 6 (160, 320, 20, 160, 40, ,10) CA04 $1,280, $1,280) HA(N158D/N224K/Q226L/ 6 of 6 (9.8) 6.1 (5) 6 of 6 ($1,280, $1,280, 640, $1,280, 4 of 6 6 of 6 (640, 640, $1280, 80, T318I)/CA04 $1,280, $1,280) $1,280, 320) rgT318I/CA04 3 of 5 (1.5)1 5.6 (3) 5 of 5 (40, 20, 20, 40, 40) 0 of 5 0 of 5 (,10, ,10, ,10, ,10, ,10) * Maximum percentage weight loss is shown. { Haemagglutination inhibition (HI) assays were carried out with homologous virus and turkey red blood cells. { One animal did not lose any body weight. 1 Two animals did not lose any body weight. 4 2 4 | N AT U R E | VO L 4 8 6 | 2 1 J U N E 2 0 1 2 ©2012 Macmillan Publishers Limited. 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APPENDIX C 87 LETTER RESEARCH a8 rgCA04 8 a 80 polykaryon formation (%) ** Wild-type HA 6 6 60 ** * N224K/Q226L HA Efficiency of Pair 1 Pair 1 4 Pair 2 4 Pair 2 40 * N158D/N224K/ Pair 3 2 Q226L HA 2 Pair 3 N158D/N224K/ 20 Q226L/T318I HA 0 0 T318I HA 0 b8 rgVN1203/CA04 8 5.4 5.5 5.6 5.7 5.8 5.9 6.0 pH 6 6 Pair 1 Pair 1 4 pH Pair 2 4 Pair 2 b 5.4 5.6 5.8 PBS 2 Pair 3 2 Pair 3 0 0 Wild-type HA c8 rg(N224K/Q226L)/CA04 8 6 6 Pair 1 Pair 1 4 N224K/ Pair 2 4 Pair 2 Q226L HA Virus titre log10(p.f.u. ml–1) Pair 3 Pair 3 2 2 0 0 N158D/ d8 HA(N158D/N224K/Q226L)/CA04 8 N224K/ Pair 1 Pair 1 Q226L HA 6 6 Pair 2 Pair 2 N158D/ 4 Pair 3 4 Pair 3 N224K/ Pair 4 Pair 4 Q226L/ 2 2 T318I HA Pair 5 Pair 5 0 Pair 6 0 Pair 6 e8 HA(N158D/N224K/Q226L/T318I)/CA04 8 T318I HA Pair 1 6 Pair 1 6 Pair 2 Pair 2 4 Pair 3 Pair 3 4 Figure 5 | Polykaryon formation by HeLa cells expressing wild-type or Pair 4 mutant HAs after acidification at low pH. a, The efficiency of polykaryon 2 Pair 4 2 Pair 5 Pair 5 formation over a pH range of 5.4–6.0 was estimated from the number of nuclei 0 Pair 6 in polykaryons divided by the total number of nuclei in the same field. The Pair 6 0 f 8 8 mean and standard deviations determined from five randomly chosen fields of rgT318I/CA04 cell culture are shown. Single asterisks indicate values significantly different 6 6 between the wild-type HA and the N224K/Q226L or N158D/N224K/Q226L Pair 1 Pair 1 HA (Tukey test; P , 0.05). The double asterisk indicates values significantly 4 Pair 2 4 Pair 2 different between the T318I HA and the N224K/Q226L or N158D/N224K/ Pair 3 Pair 3 Q226L HA (Tukey test; P , 0.05). b, Representative fields of cells expressing the 2 2 Pair 4 Pair 4 indicated HAs and exposed to pH 5.4, 5.6, or 5.8 are shown. Images were taken 0 Pair 5 0 Pair 5 at 310 magnification. Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7 Days after inoculation Days after contact wild-type fusogenic properties (that is, a threshold at pH 5.7). The HA of influenza virus undergoes a low-pH-dependent conformational Figure 4 | Respiratory droplet transmission of H5 avian–human reassortant viruses in ferrets. a–f, Groups of three, five, or six ferrets were change, which is required for fusion of the viral envelope with the inoculated intranasally with 106 p.f.u. of rgCA04 (a), rgVN1203/CA04 target membrane33. Such a conformational change to a fusion-active (b), rg(N224K/Q226L)/CA04 (c), HA(N158D/N224K/Q226L)/CA04 form can also lead to viral inactivation. Therefore, sustained and effi- (d), HA(N158D/N224K/Q226L/T318I)/CA04 (e), or rgT318I/CA04 (f). One cient human-to-human transmission of virus may require a certain day after infection, three, five, or six naive ferrets were placed in adjacent cages. level of stability of the HA protein in an acidic environment, as the pH Nasal washes were collected every other day from both inoculated (left panel) of human nasal mucosa, where human influenza viruses replicate and contact (right panel) animals for virus titration. Virus titres in organs were primarily, is approximately pH 5.5–6.5 (ref. 34). Our findings suggest determined by using a plaque assay on MDCK cells. The lower limit of that an increase in the pH threshold for fusion as a result of the N224K/ detection is indicated by the horizontal dashed line. Q226L mutations that shift the HA receptor recognition from avian- Influenza virus HA protein has membrane-fusion as well as receptor- type to human-type may reduce HA protein stability; however, the binding activity. Notably, in the three-dimensional model of influenza T318I mutation decreases the pH threshold for fusion activity, result- A virus HA, residue 318 is located proximally to the fusion peptide ing in a stable mutant HA. (Fig. 1b), which has key roles in the membrane fusion process. To Because heat treatment at neutral pH is also known to promote a assess the effect of HA mutations on low-pH-induced membrane fusogenic form of HA protein35,36 and serve as a surrogate assay for HA fusion activity, we examined the pH at which the fusion activity of stability37, we next tested whether the HA mutations described above wild-type and mutant HA was activated (Fig. 5). The wild-type HA had affect the heat stability of the HA protein. Wild-type and mutant HA a threshold for membrane fusion of pH 5.7; the N224K/Q226L and viruses were incubated at 50 uC for various times, after which the loss N158D/N224K/Q226L mutations raised the threshold for fusion to of infectivity and haemagglutination activity were determined. The .pH 5.9, whereas the T318I mutation reduced the threshold for wild-type and N224K/Q226L viruses lost most of their infectivity by fusion to pH 5.5. The N158D/N224K/Q226L/T318I mutations showed heating for 60 min (.5.5-log10 decrease in titre; Fig. 6a), whereas the 2 1 J U N E 2 0 1 2 | VO L 4 8 6 | N AT U R E | 4 2 5 ©2012 Macmillan Publishers Limited. All rights reserved

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88 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA RESEARCH LETTER a 8 N158D/N224K/Q226L and N158D/N224K/Q226L/T318I mutants VN1203/PR8 exhibited considerable tolerance to high temperature (3.9- and Virus titre log10(p.f.u. ml–1) 3.4-log10 decrease after a 60-min incubation, respectively) and the 6 N224K/Q226L T318I mutant was most resistant (only a 1.4-log10 decrease under the same conditions). In haemagglutination assays, the N224K/ 4 N158D/N224K/ Q226L mutant HA lost activity more rapidly than did the wild-type Q226L HA, and N158D/N224K/Q226L lost activity more rapidly than did the N158D/N224K/Q226L/T318I mutant (Fig. 6b). Thus, addition of the 2 N158D/N224K/ N158D mutation to the N224K/Q226L HA increased HA stability and Q226L/T318I subsequent addition of the fourth mutation, T318I, rendered the HA 0 T318I protein even more stable. Taken together, these results suggest that the 0 60 120 180 240 addition of the T318I mutation to H5 HAs that preferentially recog- Time (min) nize human-type receptors restores HA protein stability, thereby b 8 allowing a virus carrying the N158D/N224K/Q226L/T318I mutations VN1203/PR8 in HA to transmit efficiently via respiratory droplet among ferrets. In Haemagglutination titre (log2) conclusion, a fine balance of mutations affecting different functions in 6 HA (such as receptor-binding specificity and HA stability) may be N224K/Q226L critical to confer transmissibility in ferrets. 4 We next compared the pathogenicity in ferrets of H5 avian–human N158D/N224K/ reassortants with that of the pandemic H1N1 virus CA04 (Fig. 7, Q226L Supplementary Information and Supplementary Figs 9–11). The 2 N158D/N224K/ control virus, rgCA04, caused substantial body weight loss (15.1%) Q226L/T318I (Table 1 and Supplementary Fig. 9). By contrast, the four reassortant 0 T318I viruses caused only modest weight loss (,10%) in most of the animals. 0 60 120 180 240 However, no statistically significant differences in body weight loss Time (min) were found between the reassortant viruses and rgCA04. Pathological Figure 6 | Effect of heat treatment on the infectivity and haemagglutination examination revealed similar histological changes and levels of viral activity of viruses. Aliquots of a virus stock containing 128 HA units were antigens in the nasal mucosa of rgCA04-, HA(N158D/N224K/Q226L)/ incubated for the times indicated at 50 uC. a, Virus titres in heat-treated samples CA04- and HA(N158D/N224K/Q226L/T318I)/CA04-infected ferrets were determined by plaque assays on MDCK cells. b, Haemagglutination titres (Fig. 7a, b). In the rgVN1203/CA04 and rg(N224K/Q226L)/CA04 in heat-treated samples were determined by using haemagglutination assays groups, however, less tissue damage was found in the nasal mucosa with 0.5% TRBCs. Each point represents the mean 6 standard deviation from compared with the rgCA04 group on day 3 after infection (Dunnett’s triplicate experiments. test; P 5 0.0057 and 0.0175, respectively; Fig. 7b). In addition, all three a Figure 7 | Pathological analyses of H5 avian– Normal rg(N224K/Q226L)/CA04 human reassortant viruses. a, Representative histological changes in nasal turbinates from influenza-virus-infected ferrets. Three ferrets per group were infected intranasally with 106 p.f.u. of virus, and tissues were collected on day 3 after infection for pathological examination. Uninfected ferret tissues served as negative controls (normal). rgCA04 HA(N158D/N224K/Q226L)/CA04 Left panel, haematoxylin-and-eosin staining. Right panel, immunohistochemical staining for viral antigen detection (brown staining). Scale bars, 50 mm. b, Pathological severity scores in infected ferrets. To represent comprehensive histological changes, respiratory tissue slides were evaluated by scoring the pathological changes and viral antigen expression levels. The pathological scores were rgVN1203/CA04 HA(N158D/N224K/Q226L/T318I)/CA04 determined for each animal in each group (n 5 3 per group on days 3 and 6 after infection) using the following scoring system: 0, no pathological change/antigen negative; 1, affected area (,30%) or only interstitial lesion/rare viral antigens; 2, affected area (,80%, $30%)/moderate viral antigens; 3, severe lesion ($80%)/many viral b antigens. Nasal, pathological changes in the nasal mucosa; nasal Ag, viral antigens in the nasal Pathological severity score Day 3 Day 6 rgCA04 mucosa. Asterisks indicate virus pathological 3 scores significantly different from that of rgCA04 rgVN1203/CA04 (Dunnett’s test; P , 0.05). Error bars denote 2 * rg(N224K/Q226L)/CA04 standard deviation. * HA(N158D/N224K/Q226L)/CA04 1 * HA(N158D/N224K/Q226L/T318I)/CA04 0 Nasal Nasal Ag Nasal Nasal Ag 4 2 6 | N AT U R E | VO L 4 8 6 | 2 1 J U N E 2 0 1 2 ©2012 Macmillan Publishers Limited. All rights reserved

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APPENDIX C 89 LETTER RESEARCH viruses caused lung lesions (Supplementary Information and conducting surveillance in regions with circulating H5N1 viruses (for Supplementary Figs 10 and 11). example, Egypt, Indonesia, Vietnam) to recognize key residues that To assess whether current control measures may be effective against predict the pandemic potential of isolates. Rapid responses in a poten- the H5 transmissible reassortant mutant virus, we examined the reac- tial pandemic situation are essential in order to generate appropriate tivity of sera from individuals vaccinated with an H5N1 prototype vaccines and initiate other public health measures to control infection. vaccine38 against a virus possessing the N158D/N224K/Q226L/ Furthermore, our findings are of critical importance to those making T318I mutations in HA. We found that pooled human sera from public health and policy decisions. individuals immunized with this vaccine reacted with the virus posses- Our research answers a fundamental question in influenza research: sing the mutant H5 HA (N158D/N224K/Q226L/T318I) at a higher can H5-HA-possessing viruses support transmission in mammals? titre than with a wild-type H5 HA virus (VN1203/PR8; Supplementary Moreover, our findings have suggested that different mechanisms Table 6), indicating that current H5N1 vaccines would be efficacious (that is, receptor-binding specificity and HA stability) may act in con- against the H5 transmissible reassortant mutant virus. In addition, the cert for efficient transmissibility in mammals. This knowledge will H5 transmissible reassortant mutant virus (HA(N158D/N224K/ facilitate the identification of additional mutations that affect viral Q226L/T318I)/CA04) was highly susceptible to a licensed NA inhibitor, transmissibility; the monitoring of this expanded set of changes in oseltamivir (Supplementary Table 7). These experiments show that natural isolates may improve our ability to assess the pandemic poten- appropriate control measures would be available to combat the trans- tial of H5N1 viruses. Thus, although a pandemic H5N1 virus may not missible virus described in this study. possess the amino acid changes identified in our study, the findings Currently, we do not know whether the mutations that we identified described here will advance our understanding of the mechanisms and in this study that allowed the HA(N158D/N224K/Q226L/T318I)/ evolutionary pathways that contribute to avian influenza virus trans- CA04 virus to be transmissible in ferrets would also support sustained mission in mammals. human-to-human transmission. In particular, we wish to emphasize that the transmissible HA(N158D/N224K/Q226L/T318I)/CA04 virus METHODS SUMMARY possesses seven segments (all but the HA segment) from a human Viruses. All recombinant viruses were generated by using reverse genetics essen- pandemic 2009 H1N1 virus. Human-virus-characteristic amino acids tially as described previously16. All experiments with the viruses possessing the wild- type HA cleavage site were performed in an enhanced biosafety level 3 (BSL31) in these seven segments may have critically contributed to the respir- containment laboratory approved for such use by the CDC and the USDA. atory droplet transmission of the HA(N158D/N224K/Q226L/T318I)/ Infection and transmission in ferrets. Six–ten-month-old female ferrets (Triple CA04 virus in ferrets. Examples include amino acids in the PB2 F Farms) were intramuscularly anaesthetized and intranasally inoculated with polymerase protein that confer efficient replication in mammalian, 106 p.f.u. (500 ml) of virus. On days 3 and 6 after infection, ferrets were killed for but not avian, cells39–43. As the PB2 gene of the HA(N158D/N224K/ virological and pathological examinations. The virus titres in various organs were Q226L/T318I)/CA04 virus is of human virus origin, the virus determined by use of plaque assays in MDCK cells. possesses high replicative ability in mammalian cells. In contrast, most For transmission studies in ferrets, animals were housed in adjacent transmission avian virus PB2 proteins lack these human-type amino acids, although cages that prevented direct and indirect contact between animals but allowed spread one of these changes (a glutamic-acid-to-lysine mutation at position of influenza virus through the air (Showa Science; Supplementary Fig. 7). Ferrets were intranasally inoculated with 106 p.f.u. (500 ml) of virus (inoculated ferrets). 627) is found in highly pathogenic avian H5N1 viruses circulating in Twenty-four hours after infection, naive ferrets were each placed in a cage adjacent the Middle East44. As a second example, the viral NA gene may con- to an inoculated ferret (contact ferrets). To assess viral replication in the nasal tribute to viral transmissibility. The NA protein cleaves a-ketosidic turbinates, we determined viral titres in nasal washes collected from virus-inocu- linkages between a terminal sialic acid and an adjacent sugar residue, lated and contact ferrets on day 1 after inoculation or co-housing, respectively, and an activity that balances the sialic-acid-binding activity of HA. A then every other day. Animal studies were performed in accordance with Animal recent study found that a human virus NA gene was critical to confer Care and Use Committee guidelines of the University of Wisconsin-Madison. limited transmissibility to a mutant H5 avian-human reassortant Biosafety and biosecurity. All recombinant DNA protocols were approved by the virus14. In general, a human-type receptor recognizing H5 HA alone University of Wisconsin-Madison’s Institutional Biosafety Committee after risk may not be sufficient to confer transmissibility in mammals, but may assessments were conducted by the Office of Biological Safety, and by the have to act together with other human-virus-characteristic traits (in University of Tokyo’s Subcommittee on Living Modified Organisms, and, when required, by the competent minister of Japan. In addition, the University of PB2, NA, and/or other viral proteins). Therefore, at this point we Wisconsin-Madison Biosecurity Task Force regularly reviews the research pro- cannot predict whether the four mutations in the H5 HA identified gram and ongoing activities of the laboratory. The task force has a diverse skill set here would render a wholly avian H5N1 virus transmissible. and provides support in the areas of biosafety, facilities, compliance, security and Three of the residues identified here (N224, Q226 and T318) have health. Members of the Biosecurity Task Force are in frequent contact with the been strictly conserved among H5 HA proteins isolated since 2003. principal investigator and laboratory personnel to provide oversight and assure However, as H5N1 viruses continue to evolve and infect people, biosecurity. Experiments with viruses possessing the wild-type HA cleavage site receptor-binding variants of H5N1 viruses, including avian–human were performed in enhanced BSL3 containment laboratories approved for such reassortant viruses as tested here, may emerge. One of the four muta- use by the CDC and the USDA. Ferret transmission studies were conducted by three scientists with both DVM and PhD degrees who each had more than a tions we identified in our transmissible virus, the N158D mutation, minimum of 6 years of experience with highly pathogenic influenza viruses and results in loss of a glycosylation site. Many H5N1 viruses isolated in the animal studies with highly pathogenic viruses. Our staff wear powered air-puri- Middle East, Africa, Asia and Europe do not have this glycosylation fying respirators that filter the air, and disposable coveralls; they shower out on exit site. Therefore, only three nucleotide changes are needed for the HA of from the facility. The containment facilities at University of Wisconsin-Madison these viruses to support efficient transmission in ferrets. In addition, were designed to exceed standards outlined in Biosafety in Microbiological and the H5N1 viruses circulating in these geographic areas also possess a Biomedical Laboratories (5th edition; http://www.cdc.gov/biosafety/publications/ glutamic-acid-to-lysine mutation at position 627 in the PB2 protein, bmbl5/BMBL.pdf). Features of the BSL3-enhanced suites include entry/exit which promotes viral replication in certain mammals, including through a shower change room, effluent decontamination, negative air-pressure humans40,45. Therefore, these viruses may be several steps closer to laboratories, double-door autoclaves, double HEPA-filtered exhaust air, and gas those capable of efficient transmission in humans and are of concern. decontamination ports. The BSL3-Agriculture suite features include all those listed for BSL3-enhanced plus HEPA-filtered supply and double-HEPA-filtered Our study highlights the pandemic potential of viruses possessing exhaust air, double-gasketed watertight and airtight seals, airtight dampers on all an H5 HA. Although current vaccines may protect against a virus ductwork, and the structure was pressure-decay tested during commissioning. similar to that tested here, the continued evolution of H5N1 viruses The University of Wisconsin-Madison facility has a dedicated alarm system that reinforces the need to prepare and update candidate vaccines to H5 monitors all building controls and sends alarms (,500 possible alerts). viruses. The amino acid changes identified here will help individuals Redundancies and emergency resources are built-in to the facility including two 2 1 J U N E 2 0 1 2 | VO L 4 8 6 | N AT U R E | 4 2 7 ©2012 Macmillan Publishers Limited. All rights reserved

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90 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA RESEARCH LETTER air handlers, two compressors, two filters each place filters are needed, two effluent 23. Octaviani, C. P., Ozawa, M., Yamada, S., Goto, H. & Kawaoka, Y. High level of genetic sterilization tanks, two power feeds to the building, an emergency generator in case compatibility between swine-origin H1N1 and highly pathogenic avian H5N1 influenza viruses. J. Virol. 84, 10918–10922 (2010). of a power failure and other physical containment measures in the facility that 24. Li, H. et al. Isolation and characterization of H5N1 and H9N2 influenza viruses from operate without power. Biosecurity monitoring of the facility is ongoing. All pigs in China [in Chinese]. Chin. J. Prev. Vet. Med. 26, 1–6 (2004). personnel undergo Select Agent security risk assessment by the United States 25. Nidom, C. A. et al. Influenza A (H5N1) viruses from pigs, Indonesia. Emerg. Infect. Criminal Justice Information Services Division and complete rigorous biosafety, Dis. 16, 1515–1523 (2010). 26. Pasma, T. & Joseph, T. Pandemic (H1N1) 2009 infection in swine herds, Manitoba, BSL3 and Select Agent training before participating in BSL3-level experiments. Canada. Emerg. Infect. Dis. 16, 706–708 (2010). Refresher training is scheduled on a regular basis. The principal investigator 27. Pereda, A. et al. Pandemic (H1N1) 2009 outbreak on pig farm, Argentina. Emerg. participates in training sessions and emphasizes compliance to maintain safe Infect. Dis. 16, 304–307 (2010). operations and a responsible research environment. The laboratory occupational 28. Welsh, M. D. et al. Initial incursion of pandemic (H1N1) 2009 influenza A virus into health plan is in compliance with the University of Wisconsin-Madison European pigs. Vet. Rec. 166, 642–645 (2010). 29. Xu, Q., Wang, W., Cheng, X., Zengel, J. & Jin, H. Influenza H1N1 A/Solomon Island/ Occupational Health Program. Select agent virus inventory is checked monthly 3/06 virus receptor binding specificity correlates with virus pathogenicity, and submitted to the University of Wisconsin-Madison Research Compliance antigenicity, and immunogenicity in ferrets. J. Virol. 84, 4936–4945 (2010). Specialist. Virus inventory is submitted 1–2 times per year to the file holder in 30. van Riel, D. et al. Human and avian influenza viruses target different cells in the the Select Agent branch of the CDC. The research program, procedures, occu- lower respiratory tract of humans and other mammals. Am. J. Pathol. 171, pational health plan, documentation, security and facilities are reviewed annually 1215–1223 (2007). 31. Gao, Y. et al. Identification of amino acids in HA and PB2 critical for the by the University of Wisconsin-Madison Responsible Official and at regular inter- transmission of H5N1 avian influenza viruses in a mammalian host. PLoS Pathog. vals by the CDC and the Animal and Plant Health Inspection Service (APHIS) as 5, e1000709 (2009). part of the University of Wisconsin-Madison Select Agent Program. 32. Itoh, Y. et al. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 460, 1021–1025 (2009). Full Methods and any associated references are available in the online version of 33. Skehel, J. J. & Wiley, D. C. Receptor binding and membrane fusion in virus entry: the paper at www.nature.com/nature. the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000). 34. England, R. J., Homer, J. J., Knight, L. C. & Ell, S. R. Nasal pH measurement: a reliable Received 18 August 2011; accepted 9 March 2012. and repeatable parameter. Clin. Otolaryngol. Allied Sci. 24, 67–68 (1999). 35. Carr, C. M., Chaudhry, C. & Kim, P. S. Influenza hemagglutinin is spring-loaded by a Published online 2 May 2012. metastable native conformation. Proc. Natl Acad. Sci. 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Safety and influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N. Engl. J. based on species of origin. Virology 127, 361–373 (1983). Med. 354, 1343–1351 (2006). 4. Auewarakul, P. et al. An avian influenza H5N1 virus that binds to a human-type 39. Bussey, K. A., Bousse, T. L., Desmet, E. A., Kim, B. & Takimoto, T. PB2 residue 271 receptor. J. Virol. 81, 9950–9955 (2007). plays a key role in enhanced polymerase activity of influenza A viruses in 5. Gambaryan, A. et al. Evolution of the receptor binding phenotype of influenza A mammalian host cells. J. Virol. 84, 4395–4406 (2010). (H5) viruses. Virology 344, 432–438 (2006). 40. Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y. Molecular basis for high virulence of 6. Stevens, J. et al. Recent avian H5N1 viruses exhibit increased propensity for Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842 (2001). acquiring human receptor specificity. J. Mol. Biol. 381, 1382–1394 (2008). 41. Li, Z. et al. Molecular basis of replication of duck H5N1 influenza viruses in a 7. Wang, W. et al. Glycosylation at 158N of the hemagglutinin protein and receptor mammalian mouse model. J. Virol. 79, 12058–12064 (2005). binding specificity synergistically affect the antigenicity and immunogenicity of a 42. Mehle, A. & Doudna, J. A. Adaptive strategies of the influenza virus polymerase for replication in humans. Proc. Natl Acad. Sci. USA 106, 21312–21316 (2009). live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets. J. Virol. 84, 43. Yamada, S. et al. Biological and structural characterization of a host-adapting 6570–6577 (2010). amino acid in influenza virus. PLoS Pathog. 6, e1001034 (2010). 8. Watanabe, Y. et al. Acquisition of human-type receptor binding specificity by new 44. Salzberg, S. L. et al. Genome analysis linking recent European and African H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS influenza (H5N1) viruses. Emerg. Infect. Dis. 13, 713–718 (2007). Pathog. 7, e1002068 (2011). 45. Subbarao, E. K., London, W. & Murphy, B. R. A single amino acid in the PB2 gene of 9. Yamada, S. et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A virus is a determinant of host range. J. Virol. 67, 1761–1764 (1993). influenza A viruses to human-type receptors. Nature 444, 378–382 (2006). 10. Jackson, S. et al. Reassortment between avian H5N1 and human H3N2 influenza Supplementary Information is linked to the online version of the paper at viruses in ferrets: a public health risk assessment. J. Virol. 83, 8131–8140 (2009). www.nature.com/nature. 11. Maines, T. R. et al. Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc. Natl Acad. Sci. USA 103, 12121–12126 Acknowledgements The authors would like to acknowledge D. Holtzman for his contributions to the initial concept for this project and thoughtful scientific discussions. (2006). We thank M. McGregor, R. Moritz, L. Burley, K. Moore, A. Luka, J. Bettridge, N. Fujimoto 12. Maines, T. R. et al. Effect of receptor binding domain mutations on receptor binding and M. Ito for technical support, S. Watson for editing the manuscript, and the National and transmissibility of avian influenza H5N1 viruses. Virology 413, 139–147 (2011). Institute of Hygiene and Epidemiology, Hanoi, Vietnam for the A/Vietnam/1203/2004 13. Yen, H. L. et al. Inefficient transmission of H5N1 influenza viruses in a ferret contact (H5N1) virus, which was obtained from the CDC. This work was supported by the Bill & model. J. Virol. 81, 6890–6898 (2007). Melinda Gates Foundation (Grants 48339 and OPPGH5383), by a Grant-in-Aid for 14. Chen, L. M. et al. In vitro evolution of H5N1 avian influenza virus toward human- Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, type receptor specificity. Virology 422, 105–113 (2012). and Technology of Japan, by ERATO (Japan Science and Technology Agency), and by 15. Stevens, J. et al. Structure and receptor specificity of the hemagglutinin from an the National Institute of Allergy and Infectious Diseases Public Health Service Research H5N1 influenza virus. Science 312, 404–410 (2006). grants. The following reagents were obtained from the NIH Biodefense and Emerging 16. Neumann, G. et al. Generation of influenza A viruses entirely from cloned cDNAs. Infections Research Resources Repository, NIAID, NIH: polyclonal anti-monovalent Proc. Natl Acad. Sci. USA 96, 9345–9350 (1999). influenza subvirion vaccine rgA/Vietnam/1203/2004 (H5N1), (antiserum, Human), 17. Hatakeyama, S. et al. Enhanced expression of an a2,6-linked sialic acid on MDCK high tire pool, NR-4109 and low titre pool, NR-4110. cells improves isolation of human influenza viruses and evaluation of their sensitivity to a neuraminidase inhibitor. J. Clin. Microbiol. 43, 4139–4146 (2005). Author Contributions M.I., T.W., M.H., S.C.D., M.O., K.S., G.Z., A.H., H.K., S.W., C.L., S.Y., 18. Shinya, K. et al. Avian flu: influenza virus receptors in the human airway. Nature M.K., Y.S., E.A.M., G.N. and Y.K. designed the experiments; M.I., T.W., M.H., S.C.D., M.O., 440, 435–436 (2006). K.S., G.Z., A.H., H.K., S.W., C.L., S.Y. and M.K. performed the experiments; M.I., T.W., M.H., 19. Varki, A. Glycan-based interactions involving vertebrate sialic-acid-recognizing S.C.D., M.O., K.S., G.Z., A.H., H.K., S.W., C.L., E.K., S.Y., M.K., Y.S., E.A.M., G.N. and Y.K. proteins. Nature 446, 1023–1029 (2007). analysed the data; M.I., T.W., M.H., S.C.D., K.S., E.A.M., G.N. and Y.K. wrote the 20. Rogers, G. N. et al. Host-mediated selection of influenza virus receptor variants. manuscript; M.I., T.W. and M.H. contributed equally to this work. Sialic acid-a2,6Gal-specific clones of A/duck/Ukraine/1/63 revert to sialic acid- Author Information Reprints and permissions information is available at a2,3Gal-specific wild type in ovo. J. Biol. Chem. 260, 7362–7367 (1985). www.nature.com/reprints. This paper is distributed under the terms of the Creative 21. Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. X-ray structures of H5 avian and H9 Commons Attribution-Non-Commercial-Share Alike licence, and is freely available to swine influenza virus hemagglutinins bound to avian and human receptor all readers at www.nature.com/nature. The authors declare competing financial analogs. Proc. Natl Acad. Sci. USA 98, 11181–11186 (2001). interests: details accompany the full-text HTML version of the paper at 22. Cline, T. D. et al. Increased pathogenicity of a reassortant 2009 pandemic H1N1 www.nature.com/nature. Readers are welcome to comment on the online version of influenza virus containing an H5N1 hemagglutinin. J. Virol. 85, 12262–12270 this article at www.nature.com/nature. Correspondence and requests for materials (2011). should be addressed to Y.K. (kawaokay@svm.vetmed.wisc.edu). 4 2 8 | N AT U R E | VO L 4 8 6 | 2 1 J U N E 2 0 1 2 ©2012 Macmillan Publishers Limited. All rights reserved

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APPENDIX C 91 LETTER RESEARCH METHODS (Sigma) in PBS containing 0.01% H2O2 for 10 min at room temperature, and Cells. Madin–Darby canine kidney (MDCK) cells and MDCK cells overexpressing the reaction was stopped with 0.05 ml of 1 M HCl. The optical density at Siaa2,6Gal (AX4 cells17) were maintained in Eagle’s minimal essential medium 490 nm was determined in a plate reader (Infinite M1000; Tecan). (MEM) containing 5% newborn calf serum. Human embryonic kidney 293T cells Virus binding to human airway tissues. Paraffin-embedded normal human were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine trachea (US Biological) and lung (BioChain) tissue sections were deparaffinized serum (FBS). HeLa cells were maintained in MEM containing 10% FBS. All cells and rehydrated. Sections were then blocked by using 4% BSA in PBS and were maintained at 37 uC in 5% CO2. covered with virus suspensions (64 HA units in PBS) at 4 uC overnight. After Plasmid construction and reverse genetics. Plasmid constructs for viral RNA being washed four times in ice-cold PBS, the sections were incubated with primary production (pPolI)—containing the genes of the A/Vietnam/1203/2004 (H5N1; antibodies for 3 h at 4 uC. The primary antibodies used were as follows: a pool of VN1203), A/Puerto Rico/8/34 (H1N1; PR8), A/Kawasaki/173/2001 (H1N1; mouse anti-VN1203 HA monoclonal antibodies (15A3, 3G2, 7A11, 8A3, 14C5 K173) and A/California/04/2009 (H1N1; CA04) viruses flanked by the human and 18E1; Rockland); rabbit anti-K173 polyclonal antibody; rabbit anti- RNA polymerase I promoter and the mouse RNA polymerase I terminator—were surfactant protein A polyclonal antibody (Millipore); and mouse anti- constructed as described16. The multibasic amino acids at the haemagglutinin surfactant protein A monoclonal antibody (Abcam). Antibody binding was (HA) cleavage site (RERRRKKR#G) of the reassortant viruses between VN1203 detected by using an IgG secondary antibody conjugated with Alexa Fluor 488 and PR8 were changed to RETR#G by site-directed mutagenesis. All transfectant or Alexa Fluor 633 (Molecular Probes). Sections were also counterstained with viruses were generated by using reverse genetics essentially as described previ- Hoechst 33342, trihydrochloride, trihydrate (Molecular Probes). The samples ously16. Recombinant viruses were amplified in MDCK or AX417 cells and stored at were examined by using confocal laser scanning microscopy (model LSM 510; 280 uC until use. The HA segment of all viruses was sequenced to ensure the Carl Zeiss). absence of unwanted mutations. All experiments with the reassortant viruses To confirm sialic-acid-specific virus binding, tissue sections were treated, before between VN1203 and CA04 were performed in enhanced biosafety level 3 con- incubation with viruses, with Arthrobacter ureafaciens sialidase (Sigma) for 3 h at tainment laboratories approved for such use by the CDC and the USDA. 37 uC. Viruses bound to tissue were detected as described above. To introduce random mutations into the globular head of the VN1203 HA Experimental infection of ferrets. Animal studies were performed in accordance protein, a 143-amino-acid region spanning residues 120–259 (H3 numbering) was with the Animal Care and Use Committee guidelines of the University of Wisconsin- selected. This region was subjected to PCR-based random mutagenesis by use of Madison. We used 6–10-month-old female ferrets (Triple F Farms) that were the GeneMorph II kit (Stratagene) following the manufacturer’s instructions. The serologically negative by haemagglutination inhibition (HI) assay for currently targeted mutation rate (1–2 amino acid replacements per molecule) was achieved circulating human influenza viruses. Six ferrets per group were anaesthetized through optimization of the template quantity, and was confirmed by sequence intramuscularly with ketamine and xylazine (5–30 mg and 0.2–6 mg kg21 of body analysis of 48 individual clones. By using a PCR-based cloning strategy, we weight, respectively) and inoculated intranasally with 106 p.f.u. (500 ml) of viruses. inserted the mutagenized region into its respective vector containing the On days 3 and 6 after infection, three ferrets per group were killed for virological VN1203 HA gene between the human RNA polymerase I promoter and mouse and pathological examinations. The virus titres in various organs were determined RNA polymerase I terminator sequences. The composition of the plasmid library by use of plaque assays in MDCK cells. was confirmed by sequencing. The plasmid library was then used to generate an Excised tissue samples of nasal turbinates, trachea, lungs, brain, liver, spleen, influenza virus library, essentially as described16. The size of the virus library was kidney and colon from euthanized ferrets were preserved in 10% phosphate- 7 3 106 p.f.u. buffered formalin. Tissues were then trimmed and processed for paraffin Preparation of sialidase-treated TRBCs. Turkey red blood cells (TRBCs) were embedding and cut into 5-mm-thick sections. One section from each tissue sample washed three times with phosphate-buffered saline (PBS), and diluted to 20% (vol/ was stained by using a standard haematoxylin-and-eosin procedure, whereas vol) in PBS. TRBCs (1 ml) were incubated with 500 U of a2,3-sialidase from another one was processed for immunohistological staining with a mixture of Salmonella enterica serovar Typhimurium LT2 (NEB) for 20–24 h at 37 uC, two anti-influenza virus rabbit antibodies (1:2,000; R309 and anti-VN1203; both washed three times in PBS, and re-suspended in PBS or MEM containing 1% prepared in our laboratory) that react with CA04 and VN1203, respectively. bovine serum albumin (BSA) (MEM/BSA). Specific antigen–antibody reactions were visualized by using an indirect two- Haemagglutination assay. Viruses (50 ml) were serially diluted with 50 ml of PBS step dextran-polymer technique (Dako EnVision system; Dako) and 3,39 in a microtitre plate. An equal volume (that is, 50 ml) of a 0.5% (vol/vol) TRBC diaminobenzidine tetrahydrochloride staining (Dako). suspension was added to each well. The plates were kept at room temperature and Ferret transmission study. For transmission studies in ferrets, animals were haemagglutination was assessed after a 1-h incubation. housed in adjacent transmission cages that prevented direct and indirect contact Virus library screening. To select VN1203 HA variants that had acquired the between animals but allowed spread of influenza virus through the air (Showa ability to recognize human-type receptors, three parallel experiments were carried Science; Supplementary Fig. 7). Three, five, or six ferrets were inoculated out, each with 0.7 3 106 viruses. The virus library was first incubated with 0.1 ml of intranasally with 106 p.f.u. (500 ml) of virus (inoculated ferrets). Twenty-four hours 10% (vol/vol) a2,3-sialidase-treated TRBCs for 10 min at 4 uC. After this incuba- after infection, three, five, or six naive ferrets were each placed in a cage adjacent to tion, the TRBCs and bound viruses were pelleted at 1,000 r.p.m. for 1 min, and the an inoculated ferret (contact ferrets). The ferrets were monitored for changes in pellets then washed ten times in MEM/BSA containing 313 mM NaCl. Bound viruses were eluted by incubation at 37 uC for 30 min and then diluted to approxi- body weight and the presence of clinical signs. To assess viral replication in nasal mately 0.5 virus per well (determined by virus titration in a pilot study). Individual turbinates, we determined viral titres in nasal washes collected from virus- viruses were then amplified in AX4 cells, which overexpress Siaa2,6Gal17. inoculated and contact ferrets on day 1 after inoculation or co-housing, respect- Individual viruses were re-screened by using haemagglutination assays with ively, and then every other day. a2,3-sialidase-treated TRBCs. Serological tests. Serum samples were collected between days 14 and 20 after infec- Solid-phase binding assay. Viruses were grown in MDCK cells, clarified by low- tion, treated with receptor-destroying enzyme, heat-inactivated at 56 uC for 30 min, speed centrifugation, laid over a cushion of 30% sucrose in PBS, and ultracentri- and tested by use of an HI assay with 0.5% TRBCs (http://www.wpro.who.int/entity/ fuged at 25,000 r.p.m. for 2 h at 4 uC. Virus stocks were aliquoted and stored at emerging_diseases/documents/docs/manualonanimalaidiagnosisandsurveillance. 280 uC. Virus concentrations were determined by using haemagglutination assays pdf). Viruses bearing homologous HA were used as antigens for the HI tests. with 0.5% (vol/vol) TRBCs. The direct receptor-binding capacity of viruses was Polykaryon formation representing membrane fusion activity. Monolayers of examined by use of a solid-phase binding assay as previously described9. Microtitre HeLa cells grown in 12-well plates were transfected with the protein expression plates (Nunc) were incubated with the sodium salts of sialylglycopolymers (poly- vector pCAGGS46 encoding wild-type or mutant HA. At 24 h after transfection, L-glutamic acid backbones containing N-acetylneuraminic acid linked to galactose cells transiently expressing HA protein were treated with trypsin (1 mg ml21) in through either an a2,3 (Neu5Aca2,3Galb1,4GlcNAcb1-pAP) or an a2,6 MEM containing 0.3% BSA for 30 min at 37 uC to cleave the HA into its HA1 and (Neu5Aca2,6Galb1,4GlcNAcb1-pAP) bond) in PBS at 4 uC overnight. After the HA2 subunits. Polykaryon formation was induced by exposing the cells to low-pH glycopolymer solution was removed, the plates were blocked with 0.15 ml of PBS buffer (145 mM NaCl, 20 mM sodium citrate (pH 6.0–5.4)) for 2 min at 37 uC. containing 4% BSA at room temperature for 1 h. After four successive washes with After this exposure, the low-pH buffer was replaced with MEM containing 10% ice-cold PBS, the plates were incubated in a solution containing influenza virus (8– FBS and the cells were incubated for 3 h at 37 uC. The cells were then fixed with 32 HA units in PBS) at 4 uC overnight. After washing as described above, the plates methanol and stained with Giemsa’s solution and photographed with a digital were incubated for 2 h at 4 uC with rabbit polyclonal antiserum to either K173 or camera mounted on an inverted microscope (Nikon, Eclipse Ti). For quantitative VN1203 virus. The plates were then washed again as before and incubated with analyses, cell nuclei were counted in five randomly chosen fields of cell culture. horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antiserum for 2 h Polykaryon formation activity was calculated from the number of nuclei in at 4 uC. After washing, the plates were incubated with O-phenylenediamine polykaryons divided by the total number of nuclei in the same field. ©2012 Macmillan Publishers Limited. All rights reserved

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92 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA RESEARCH LETTER Thermostability. Viruses (128 HA units in PBS) were incubated for the times Statistical analysis. All statistical analyses were performed using JMP 9.0.0 (SAS indicated at 50 uC. Subsequently, infectivity and haemagglutination activity were Institute Inc.). The statistical significance of differences between rgCA04 and H5 determined by use of plaque assays in MDCK cells and haemagglutination assays avian/human reassortant viruses was determined by using a Dunnett’s test. using 0.5% TRBCs, respectively. Comparisons of polykaryon formation between wild-type and mutant HAs were Neuraminidase (NA) inhibition assay. To assess the sensitivity of viruses to done using Tukey’s test. P values of ,0.05 were considered significant. the NA inhibitor oseltamivir, NA inhibition assays were performed as described 46. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression previously32. transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991). ©2012 Macmillan Publishers Limited. All rights reserved

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APPENDIX C 93 H5N1 second small protein, PB1-F2, which has been REPORT implicated in the induction of cell death (4, 5). Segments 4 and 6 encode the viral surface glyco- Airborne Transmission of Influenza proteins hemagglutinin (HA) and neuraminidase (NA), respectively. HA is responsible for binding A/H5N1 Virus Between Ferrets to sialic acids (SAs), the viral receptors on host cells, and for fusion of the viral and host cell membranes upon endocytosis. NA is a sialidase, Sander Herfst,1 Eefje J. A. Schrauwen,1 Martin Linster,1 Salin Chutinimitkul,1 Emmie de Wit,1* responsible for cleaving SAs from host cells and Vincent J. Munster,1* Erin M. Sorrell,1 Theo M. Bestebroer,1 David F. Burke,2 Derek J. Smith,1,2,3 virus particles. Segment 5 codes for the nucleo- Guus F. Rimmelzwaan,1 Albert D. M. E. Osterhaus,1 Ron A. M. Fouchier1† capsid protein (NP) that binds to viral RNA and, Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far together with the polymerase proteins, forms the has not acquired the ability to be transmitted by aerosol or respiratory droplet (“airborne transmission”) ribonucleoprotein complexes (RNPs). Segment 7 between humans. To address the concern that the virus could acquire this ability under natural conditions, codes for the viral matrix structural protein M1 we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in and the ion-channel protein M2 that is incorpo- ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately rated in the viral membrane. Segment 8 encodes becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the nonstructural protein NS1 and the nucleic- the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein export protein (NEP) previously known as NS2. hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present NS1 is an antagonist of host innate immune re- in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir sponses and interferes with host gene expression, and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza whereas NEP is involved in the nuclear export viruses can acquire the capacity for airborne transmission between mammals without recombination in an of RNPs into the cytoplasm before virus assem- intermediate host and therefore constitute a risk for human pandemic influenza. bly (2, 3). Influenza A viruses show pronounced genetic I nfluenza A viruses have been isolated from riiformes (gulls, terns, and waders) are thought to variation of the surface glycoproteins HA and many host species, including humans, pigs, form the virus reservoir in nature (1). Influenza A NA (1). Consequently, the viruses are classified horses, dogs, marine mammals, and a wide viruses belong to the family Orthomyxoviridae; based on the antigenic variation of the HA and range of domestic birds, yet wild birds in the orders these viruses have an RNA genome consisting of NA proteins. To date, 16 major antigenic variants Anseriformes (ducks, geese, and swans) and Charad- eight gene segments (2, 3). Segments 1 to 3 en- of HA and 9 of NA have been recognized in wild code the polymerase proteins: basic polymerase birds and are found in numerous combinations 1 Department of Virology, Erasmus Medical Center, Rotterdam, designated as virus subtypes (for instance, H1N1, 2 (PB2), basic polymerase 1 (PB1), and acidic The Netherlands. 2Department of Zoology, University of Cam- H5N1, H7N7, and H16N3), which are used in bridge, Cambridge, UK. 3Fogarty International Center, National polymerase (PA), respectively. These proteins Institutes of Health (NIH), Bethesda, MD 20892, USA. form the RNA-dependent RNA polymerase com- influenza A virus classification and nomenclature †To whom correspondence should be addressed. E-mail: plex responsible for transcription and replication (1, 6). This classification system is biologically r.fouchier@erasmusmc.nl of the viral genome. Segment 2 also encodes a relevant, as natural host antibodies that recognize A B C D log 10 TCID 50 / ml 6 6! A/H5N1 6! A/H5N1 N182K 6! A/H5N1 Q222L G224S 6! A/H5N1 N182K Q222L G224S 5! 5! 5! 5! 4 nose 4! 4! 4! 4! 3! 3! 3! 3! 2 2! 2! 2! 2! 1! 1! 1! 1! 0 0! 0! 0! 0! log 10 TCID 50 / ml 6 6! 6! 6! 6 6! 5! 5! 5! 5! 5 throat 4 4! 4! 4! 4! 4 3! 3! 3! 3! 3 2 2! 2! 2! 2! 2 1! 1! 1! 1! 1 0 0! 0! 0! 0 0! day 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 log 10 TCID 50 / g tissue 8 8 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 6 6 6 6 6 6 6 6 6 tissue 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 NT T L NT T L NT T L NT T L NT T L NT T L NT T L NT T L 3 dpi 7 dpi 3 dpi 7 dpi 3 dpi 7 dpi 3 dpi 7 dpi Fig. 1. In experiment 1, we inoculated groups of six ferrets intranasally with shedding from the URT as determined by virus titers in nasal and throat swabs 1 × 106 TCID50 of (A) influenza A/H5N1wildtype virus and the three mutants (B) was highest in A/H5N1wildtype-inoculated animals. The mutant that yielded the A/H5N1HA N182K, (C) A/H5N1HA Q222L,G224S, and (D) A/H5N1HA N182K,Q222L,G224S. highest virus titers during the 7-day period was A/H5N1HA Q222L,G224S, but titers Three animals were euthanized at day 3 for tissue sampling and at day 7, when were ~1 log lower than for the A/H5N1wildtype-inoculated animals. In the first this experiment was stopped. Virus titers were measured daily in nose swabs 3 days, when six animals per group were present, no significant differences (top) and throat swabs (middle) and also on 3 and 7 dpi in respiratory tract were observed between A/H5N1HA N182K- and A/H5N1HA Q222L,G224S-inoculated tissues (bottom) of individual ferrets. Virus titers in swabs and nasal turbinates animals, as calculated by comparing the viral titer (Mann-Whitney test, P = (NT), trachea (T), and lungs (L) were determined by end-point titration in MDCK 0.589 and 0.818 for nose and throat titers, respectively). (Bottom row) No cells. [One animal inoculated with A/H5N1HA N182K,Q222L,G224S died at 1 dpi due marked differences in virus titers in respiratory tissues were observed between to circumstances not related to the experiment (D).] (Top two rows) Virus the four groups. Each bar color denotes a single animal. 1534 22 JUNE 2012 VOL 336 SCIENCE www.sciencemag.org

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94 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA SPECIALSECTION Fig. 2. Experiment 3, virus passaging in ferrets (P1 to P10, passages 1 to 10). 3 to 6 were performed in the same way. From passage six onward, nasal Because no airborne transmission was observed in experiment 2, A/H5N1wildtype washes (NW) were collected at 3 dpi in addition to the nasal swabs. To this end, and A/H5N1HA Q222L,G224S PB2 E627K were serially passaged in ferrets to allow 1 ml of PBS was delivered drop wise into the nostrils of the ferrets, thereby adaptation for efficient replication in mammals. Each virus was inoculated inducing sneezing. Approximately 200 ml of the sneeze was collected in a Petri intranasally with 1 × 106 TCID50 in one ferret (2 × 250 ml, divided over both dish, and PBS was added to a final volume of 2 ml. For passages 7 through 10, nostrils). Nose and throat swabs were collected daily. Animals were euthanized the nasal-wash sample was used for the passages in ferrets. The passage-10 at 4 dpi, and nasal turbinates and lungs were collected. Nasal turbinates were nasal washes were subsequently used for sequence analyses and transmission homogenized in virus-transport medium, and this homogenate was used to experiments to be described in experiment 4. For details, see the supple- inoculate the next ferret, resulting in passage 2 (fig. S6). Subsequent passages mentary materials. one HA or NA subtype will generally not cross- Fig. 3. Virus titers in react with other HA and NA subtypes. (A) the nasal turbinates On the basis of their virulence in chickens, collected at day 4 and influenza A viruses of the H5 and H7 subtypes (B) nose swabs collected can be further classified into highly pathogenic daily until day 4, from avian influenza (HPAI) and low-pathogenic avian ferrets inoculated with influenza (LPAI) viruses. Viruses of subtypes A/H5N1wildtype (blue) and H1 to H4, H6, and H8 to H16 are LPAI viruses. A/H5N1HA Q222L,G224S PB2 E627K The vast majority of H5 and H7 influenza A vi- (red) throughout the 10 serial passages described ruses are also of the LPAI phenotype. HPAI vi- in Fig. 2. Virus titers were determined by end-point titration in ruses are generally thought to arise in poultry MDCK cells. After inoculation with A/H5N1wildtype, virus titers after domestic birds become infected by LPAI H5 in the nasal turbinates were variable but high, ranging from and H7 viruses from the wild-bird reservoir (7, 8). 1.6 × 105 to 7.9 × 106 TCID50/gram tissue (A), with no further The HA protein of influenza A viruses is initial- increase observed with repeated passage. After inoculation ly synthesized as a single polypeptide precursor with A/H5N1HA Q222L,G224S PB2 E627K, virus titers in nasal tur- (HA0), which is cleaved into HA1 and HA2 sub- binates averaged 1.6 × 104 in the first three passages, 2.5 × units by trypsin-like proteases in the host cell. 105 in passages four to seven, and 6.3 × 105 TCID50/gram tissue The switch from LPAI to HPAI virus phenotype in the last three passages, suggestive of improved replication and occurs upon the introduction of basic amino acid virus adaptation. A similar pattern of adaptation was observed residues into the HA0 cleavage site, also known in the virus titers in the nose swabs of animals inoculated with as the multibasic cleavage site (MBCS). The A/H5N1HA Q222L,G224S PB2 E627K (B). These titers also increased MBCS in HA can be cleaved by ubiquitously ex- during the successive passages, with peak virus shedding of 1 × pressed host proteases; this cleavage facilitates 105 TCID50 at 2 dpi after 10 passages. Altogether, these data systemic virus replication and results in mortality indicate that A/H5N1HA Q222L,G224S PB2 E627K adapted to more of up to 100% in poultry (9, 10). efficient replication in the ferret URT upon repeated passage, Since the late 1990s, HPAI A/H5N1 viruses with evidence for such adaptation by passage number 4. In contrast, have devastated the poultry industry of numerous analyses of the virus titers in the nose swabs of the ferrets collected at 1 to 4 dpi throughout the 10 serial countries in the Eastern Hemisphere. To date, passages with A/H5N1wildtype revealed no changes in patterns of virus shedding. Asterisks indicate that a nose A/H5N1 has spread from Asia to Europe, Africa, wash was collected before the nose swab was taken, which may influence the virus titer that was detected. and the Middle East, resulting in the death of hundreds of millions of domestic birds. In Hong (12). Although limited A/H5N1 virus transmission droplets among mammals, including humans, to Kong in 1997, the first human deaths directly between persons in close contact has been re- trigger a future pandemic is a key question for attributable to avian A/H5N1 virus were recorded ported, sustained human-to-human transmission pandemic preparedness. Although our knowledge (11). Since 2003, more than 600 laboratory- of HPAI A/H5N1 virus has not been detected of viral traits necessary for host switching and confirmed cases of HPAI A/H5N1 virus infections (13–15). Whether this virus may acquire the abil- virulence has increased substantially in recent in humans have been reported from 15 countries ity to be transmitted via aerosols or respiratory years (16, 17), the factors that determine airborne www.sciencemag.org SCIENCE VOL 336 22 JUNE 2012 1535

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APPENDIX C 95 H5N1 virus passage transmission 1 transmission 2 transmission 3 F1 P0 F2 F5 F7 P10 F3 F6 F8 Fig. 4. Summary of the substitutions detected upon serial passage and airborne F4 transmission of A/H5N1HA Q222L,G224S PB2 E627K virus in ferrets. The eight influenza virus gene segments and substitutions are drawn approximately to scale (top to bottom: PB2, PB1, PA, HA, NP, NA, M, NS). Viruses shown in blue, orange, and red represent the initial recombinant A/H5N1HA Q222L,G224S PB2 E627K virus (P0), ferret passage-10 virus (P10), and P10 virus after airborne transmission to recipient ferrets, respectively. Viruses shown in gray indicate that virus was not transmitted to the recipient ferret. First, we tested whether airborne-transmissible viruses were present in the heterogeneous virus population of ferret P10. We inoculated four donor ferrets intranasally, which were then housed in transmission cages and paired with four recipient ferrets. Transmissible viruses were isolated from three out of four recipient ferrets (F1 to F3). Next, we took a throat-swab sample from F2 (this sample contained the highest virus titer among the positive recipient ferrets), and this sample was used to inoculate two more donor ferrets intranasally. In a transmission experiment, these donors infected two recipient ferrets via airborne transmission (F5 and F6). Virus isolated from F5 was passaged once in MDCK cells and was subsequently used in a third transmission experiment in which two intranasally inoculated donor ferrets transmitted the virus to one of two recipient ferrets (F7). The genetic composition of the viral quasi-species present in the nasal wash of ferret P10 was determined by sequence analysis using the 454/Roche GS-FLX sequencing platform. Conventional Sanger sequencing was used to determine the consensus sequence in one high-titer nasal- or throat-swab sample for each ferret. Thick and thin black vertical bars indicate amino acid and nucleotide substitutions, respectively; substitutions introduced by reverse genetics are shown in yellow; substitutions detected in passage 10 and all subsequent transmissions are shown in green. transmission of influenza viruses among mam- events between A/H5N1 and seasonal human human influenza viruses have been detected in mals, a trait necessary for a virus to become pan- influenza viruses do not yield viruses that are nature and because our goal was to understand demic, have remained largely unknown (18–21). readily transmitted between ferrets (18–20, 23). the biological properties needed for an influ- Therefore, investigations of routes of influenza In our work, we investigated whether A/H5N1 enza virus to become airborne transmissible virus transmission between animals and on the virus could change its transmissibility charac- in mammals, we decided to use the complete determinants of airborne transmission are high teristics without any requirement for reassort- A/Indonesia/5/2005 virus that was isolated from on the influenza research agenda. ment. a human case of HPAI A/H5N1 infection. The viruses that caused the major pandemics We chose influenza virus A/Indonesia/5/2005 We chose the ferret (Mustela putorius furo) as of the past century emerged upon reassortment for our study because the incidence of human the animal model for our studies. Ferrets have (that is, genetic mixing) of animal and human in- A/H5N1 virus infections and fatalities in Indo- been used in influenza research since 1933 be- fluenza viruses (22). However, given that viruses nesia remains fairly high (12), and there are cause they are susceptible to infection with human from only four pandemics are available for analy- concerns that this virus could acquire molecular and avian influenza viruses (24). After infection ses, we cannot exclude the possibility that a future characteristics that would allow it to become with human influenza A virus, ferrets develop pandemic may be triggered by a wholly avian more readily transmissible between humans and respiratory disease and lung pathology similar to virus without the requirement of reassortment. initiate a pandemic. Because no reassortants be- that observed in humans. Ferrets can also trans- Several studies have shown that reassortment tween A/H5N1 viruses and seasonal or pandemic mit human influenza viruses to other ferrets that 1536 22 JUNE 2012 VOL 336 SCIENCE www.sciencemag.org

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96 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA SPECIALSECTION serve as sentinels with or without direct contact Fig. 5. Airborne transmission of (fig. S1) (25–27). A/H5N1 viruses in ferrets. Trans- Host restriction of replication and trans- mission experiments are shown mission of influenza A viruses is partly determined for A/H5N1wildtype (A and B) and by specific SA receptors on the surface of sus- A/H5N1HA Q222L,G224S PB2 E627K ceptible cells. The affinity of influenza viruses (C and D) after 10 passages (P10) for these receptors varies according to the in ferrets. Two or four ferrets were species from which they are isolated. Influenza inoculated intranasally with nasal- viruses of avian origin preferentially bind to wash samples collected from a-2,3–linked SA receptors, whereas human P10 virus of A/H5N1wildtype and influenza viruses recognize a-2,6–linked SA re- A/H5N1HA Q222L,G224S PB2 E627K, respectively, and housed individu- ceptors. The receptor distribution in ferrets re- ally in transmission cages (A and sembles that of humans in that the a-2,6–linked C). A naïve recipient ferret was added SA receptors are predominantly present in the to each transmission cage adjacent upper respiratory tract (URT), and the a-2,3– to a donor ferret at 1dpi (B and D). linked SA receptors are mainly present in the Virus titers in throat (black bars) lower respiratory tract. In chickens and other and nose swabs (white bars) were birds, a-2,3–linked SAs predominate, but both determined by end-point titration a-2,3–linked and a-2,6–linked SA are present in MDCK cells. Geometric mean titers throughout the respiratory and enteric tracts and SDs (error bars) of positive samples are shown. The number of animals infected via airborne transmission (fig. S2) (28). The differences in receptor distri- is indicated in (D) for each time point after exposure; the drop from three animals infected at day 7 to one bution between humans and avian species are animal at day 9 and no animals at day 11 is explained by the fact that the animals that became infected via thought to determine the host restriction of in- airborne transmission had cleared the virus by the end of the experiment and, therefore, detectable amounts fluenza A viruses. A switch in receptor spec- of virus were no longer present. The dotted lines indicate the lower limit of virus detection. ificity from avian a-2,3–SA to human a-2,6–SA receptors, which can be acquired by specific mu- tations in the receptor binding site (RBS) of the HA, is expected to be necessary for an avian vi- rus to become transmissible and, thus, gain the potential to become pandemic in humans. Besides a switch in receptor specificity to facilitate infection of cells in the URT, increased virus production in the URT and efficient release of virus particles from the respiratory tract to yield airborne virus may also be required (22). Such traits are likely to be determined by the viral surface glycoproteins and the proteins that form the viral polymerase complex. Amino acid substitutions in the polymerase proteins have al- ready been shown to be major determinants of host range and transmission, including for pan- Fig. 6. Comparison of airborne transmission of experimental passaged A/H5N1 and 2009 pandemic demic influenza viruses (29–31). Whereas avian A/H1N1 viruses in individual ferrets. A throat-swab sample from ferret F2 at 7 days postexposure (dpe) viruses, in principle, replicate at temperatures (Fig. 5D) was used for the transmission experiments shown in (A) and (B), and a virus isolate obtained around 41°C (the temperature in the intestinal from a nose swab collected from ferret F5 at 7 dpi (Fig. 6A) was used for the experiments in (C) and (D). tract of birds), for replication in humans the vi- For comparison, published data on transmission of 2009 pandemic A/H1N1 virus between ferrets is shown ruses need to adapt to 33°C (the temperature of in (E) to (H) (27). Data for individual transmission experiments is shown in each panel, with virus shedding in inoculated and airborne virus–exposed animals shown as lines and bars, respectively. For the trans- the human URT). The amino acid substitution mission experiments with airborne-transmissible A/H5N1 (A to D), nose or throat swabs were not collected at 2 dpi Glu627→Lys627 (E627K) in the polymerase com- and 2 dpe. White circles and bars represent shedding from the nose; black circles and bars represent shedding plex protein PB2 has been associated with in- from the throat. The asterisk indicates the inoculated animal that died 6 days after intranasal inoculation. creased virus replication in mammalian cells at such lower temperatures (16, 17, 32). In addition, when newly formed virus par- Human-to-human transmission of influenza sols. However, it is generally accepted that for ticles bud from the host cell membrane after viruses can occur through direct contact, indirect infectious particles with a diameter of 5 mm or virus replication, the NA present on the virus contact via fomites (contaminated environmental less, transmission occurs via aerosols. Because membrane facilitates the release of particles. surfaces), and/or airborne transmission via small we did not measure particle size during our ex- For A/H5N1, this process is rather inefficient, aerosols or large respiratory droplets. The pan- periments, we will use the term “airborne trans- and released particles tend to form virus ag- demic and epidemic influenza viruses that have mission” throughout this Report. gregates (22). Therefore, a balance between the circulated in humans throughout the past century Biosafety and biosecurity concerns have re- properties endowed by HA and NA may be were all transmitted via the airborne route, in mained foremost in our planning for this research required to generate single particles. These estab- contrast to many other respiratory viruses that are program. The details are explained in the supple- lished effects were thus used as the basis for exclusively transmitted via contact. There is no mentary materials and are summarized here: The the initial substitutions chosen in the current exact particle size cut-off at which transmission enhanced Animal Biosafety Laboratory level 3 study. changes from exclusively large droplets to aero- (ABSL3+) facility at Erasmus Medical Center www.sciencemag.org SCIENCE VOL 336 22 JUNE 2012 1537

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APPENDIX C 97 H5N1 Table 1. Lethality of WT and airborne-transmissible A/H5N1 virus in ferrets upon inoculation via tant A/H5N1 virus with receptor specificity for different routes. n, number of animals; N.A., not applicable. a-2,6–linked SA shed at high titers from the URT of ferrets. Therefore, we used the QuickChange Dead or moribund Day of death multisite-directed mutagenesis kit (Agilent Tech- Inoculation route Virus (no. dead/no. tested) postinoculation (no.) nologies, Amstelveen, the Netherlands) to intro- Intratracheal A/H5N1wildtype 6/6* 2 (n = 2), 3 (n = 4) duce amino acid substitutions N182K, Q222L/ A/H5N1/F5 6/6 3 (n = 6) G224S, or N182K/Q222L/G224S in the HA of Intranasal A/H5N1wildtype/P10 2/2† 6 (n = 2) wild-type (WT) A/Indonesia/5/2005, resulting A/H5N1HA Q222L,G224S PB2 E627K/P10 0/4 N.A. in A/H5N1HA N182K, A/H5N1HA Q222L,G224S, and A/H5N1/F2 0/2 N.A. A/H5N1HA N182K,Q222L,G224S. Experimental A/H5N1/F5 1/2 6 (n = 1) details for experiments 1 to 9 are provided in Airborne A/H5N1wildtype N.A. N.A. the supplementary materials (25). For experi- A/H5N1HA Q222L,G224S PB2 E627K/P10 0/3 N.A. ment 1, we inoculated these mutant viruses and A/H5N1/F2 0/2 N.A. the A/H5N1wildtype virus intranasally into groups A/H5N1/F5 0/1 N.A. of six ferrets for each virus (fig. S3). Throat and *These data refer to a published study (45). †These ferrets were inoculated with P10 H5N1wildtype virus, but data are nasal swabs were collected daily, and virus titers consistent with previous studies that used larger groups of animals inoculated with the original strain (39, 40). were determined by end-point dilution in Madin Darby canine kidney (MDCK) cells to quantify Table 2. Receptor specificity of the different mutant A/H5N1 viruses, as determined by a modified TRBC virus shedding from the ferret URT. Three an- hemagglutination assay. Introduction of Q222L and G224S in the A/H5N1 HA resulted in a receptor binding imals were euthanized after day 3 to enable tissue preference switch from the avian a-2,3– to the human a-2,6–linked SA receptor. Subsequent substitution of sample collection. All remaining animals were H103Y and T156A resulted in an increased affinity for a-2,3– and a-2,6–linked SA, in agreement with euthanized by day 7 when the same tissue samples glycan array studies (51). For details, see supplementary experiment 9. HAU, hemagglutination units. were taken. Virus titers were determined in the nasal turbinates, trachea, and lungs collected post- HA titer (HAU/50 ml) Virus Subtype mortem from the euthanized ferrets. Throughout TRBC a-2,3–linked TRBC a-2,6–linked TRBC the duration of experiment 1, ferrets inoculated in- A/Netherlands/213/03 H3N2 64 0 64 tranasally with A/H5N1wildtype virus produced A/Vietnam/1194/04 H5N1 64 64 0 high titers in nose and throat swabs—up to 10 times A/H5N1PB2 E627K H5N1 64 16 0 more than A/H5N1HA Q222L,G224S, which yielded A/H5N1HA H103Y,T156A PB2 E627K H5N1 64 48 0 the highest virus titers of all three mutants during A/H5N1HA Q222L,G224S PB2 E627K H5N1 64 0 24 the 7-day period (Fig. 1). However, no significant A/H5N1HA H103Y,T156A,Q222L,G224S PB2 E627K H5N1 64 4 32 difference was observed between the virus shed- ding of ferrets inoculated with A/H5N1HA Q222L, G224S or A/H5N1HA N182K during the first 3 days (MC) Rotterdam, the Netherlands, was constructed and A/H5N1 influenza vaccines (25). For emer- when six animals per group were present. Thus, for the specific purpose of containing pathogenic gency purposes, Erasmus MC holds supplies of of the viruses with specificity for a-2,6–linked SA, and transmissible influenza viruses and other oseltamivir and has quarantine hospital rooms. A/H5N1HA Q222L,G224S yielded the highest virus pathogens of concern. The facility consists of a Using a combination of targeted mutagenesis titers in the ferret URT (Fig. 1). negatively pressurized laboratory with an inter- followed by serial virus passage in ferrets, we As described above, amino acid substitution lock room. All in vivo and in vitro experimen- investigated whether A/H5N1 virus can acquire E627K in PB2 is one of the most consistent host- tal work is carried out in negatively pressurized mutations that would increase the risk of mam- range determinants of influenza viruses (29–31). class 3 isolators or class 3 biosafety cabinets, re- malian transmission (34). We have previous- For experiment 2 (fig. S4), we introduced E627K spectively. The facility is secured by procedures ly shown that several amino acid substitutions into the PB2 gene of A/Indonesia/5/2005 by recognized as appropriate by the institutional in the RBS of the HA surface glycoprotein of site-directed mutagenesis and produced the re- biosafety officers and facility management at A/Indonesia/5/2005 change the binding pref- combinant virus A/H5N1HA Q222L,G224S PB2 E627K. Erasmus MC, as well as Dutch and U.S. gov- erence from the avian a-2,3–linked SA recep- The introduction of E627K in PB2 did not sig- ernment inspectors. tors to the human a-2,6–linked SA receptors nificantly affect virus shedding in ferrets, because Before and during the research, biosafety (35). A/Indonesia/5/2005 virus with amino virus titers in the URT were similar to those seen officers of Erasmus MC and inspectors from acid substitutions N182K, Q222L/G224S, or in A/H5N1HA Q222L,G224S-inoculated animals the Dutch government, as well as from the U.S. N182K/Q222L/G224S (numbers refer to amino [up to 1 × 104 50% tissue culture infectious Centers for Disease Control and Prevention, acid positions in the mature H5 HA protein; doses (TCID50)] (Mann-Whitney U rank-sum test, approved the facilities and procedures. Explicit N, Asn; Q, Gln; L, Leu; G, Gly; S, Ser) in HA P = 0.476) (Fig. 1 and fig. S5). When four naïve permits for research on genetically modified display attachment patterns similar to those of ferrets were housed in cages adjacent to those airborne-transmissible A/H5N1 virus were ob- human viruses to cells of the respiratory tract of with four inoculated animals to test for air- tained from the Dutch government. The research ferrets and humans (35). Of these changes, we borne transmission as described previously (27), was performed strictly in accordance with the know that together, Q222L and G224S switch the A/H5N1HA Q222L,G224S PB2 E627K was not trans- Dutch Code of Conduct for Biosecurity (33). All receptor binding specificity of H2 and H3 sub- mitted (fig. S5). personnel were instructed and trained extensive- type influenza viruses, as this switch contributed Because the mutant virus harboring the ly for working in the ABSL3+ facility, handling to the emergence of the 1957 and 1968 pan- E627K mutation in PB2 and Q222L and G224S (highly pathogenic) influenza virus, and control- demics (36). N182K has been found in a human in HA did not transmit in experiment 2, we de- ling incidents (such as spills). To further prevent case of A/H5N1 virus infection (37). signed an experiment to force the virus to adapt occupational risks, research personnel used pro- Our experimental rationale to obtain trans- to replication in the mammalian respiratory tective equipment and were offered seasonal missible A/H5N1 viruses was to select a mu- tract and to select virus variants by repeated 1538 22 JUNE 2012 VOL 336 SCIENCE www.sciencemag.org

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98 PERSPECTIVES ON RESEARCH WITH H5N1 AVIAN INFLUENZA SPECIALSECTION passage (10 passages in total) of the constructed of ferrets after 10 passages of A/H5N1wildtype sion experiment, was subsequently used for intra- A/H5N1HA Q222L,G224S PB2 E627K virus and and A/H5N1HA Q222L,G224S PB2 E627K was deter- nasal inoculation of two additional donor ferrets. A/H5N1wildtype virus in the ferret URT (Fig. 2 mined by sequence analysis using the 454/Roche Both of these animals, when placed in the trans- and fig. S6). In experiment 3, one ferret was in- GS-FLX sequencing platform (Roche, Woerden, mission cage setup (fig. S1), again transmitted oculated intranasally with A/H5N1wildtype and the Netherlands) (tables S1 and S2). The the virus to the recipient ferrets (F5 and F6) one ferret with A/H5N1HA Q222L,G224S PB2 E627K. mutations introduced in A/H5N1HA Q222L,G224S (Fig. 6, A and B). A virus isolate was obtained Throat and nose swabs were collected daily PB2 E627K by reverse genetics remained present after inoculation of MDCK cells with a nose swab from live animals until 4 days postinoculation in the virus population after 10 consecutive collected from ferret F5 at 7 dpi. The virus from (dpi), at which time the animals were euthanized passages at a frequency >99.5% (Fig. 4 and F5 was inoculated intranasally into two more to collect samples from nasal turbinates and lungs. table S1). Numerous additional nucleotide sub- donor ferrets. One day later, these animals were The nasal turbinates were homogenized in 3 ml stitutions were detected in all viral gene segments paired with two recipient ferrets (F7 and F8) in of virus-transport medium, tissue debris was pel- of A/H5N1wildtype and A/H5N1HA Q222L,G224S PB2 E627K transmission cages, one of which (F7) subsequent- leted by centrifugation, and 0.5 ml of the super- after passaging, except in segment 7 (tables S1 ly became infected (Fig. 6, C and D). natant was subsequently used to inoculate the and S2). Of the 30 nucleotide substitutions selec- We used conventional Sanger sequencing to next ferret intranasally (passage 2). This proce- ted during serial passage, 53% resulted in amino determine the consensus genome sequences of dure was repeated until passage 6. acid substitutions. The only amino acid substi- viruses recovered from the six ferrets (F1 to F3 From passage 6 onward, in addition to the tution detected upon repeated passage of both and F5 to F7) that acquired virus via airborne samples described above, a nasal wash was also A/H5N1wildtype and A/H5N1HA Q222L,G224S PB2 E627K transmission (Fig. 4 and table S3). All six sam- collected at 3 dpi. To this end, 1 ml of phosphate- was T156A (T, Thr; A, Ala) in HA. This sub- ples still harbored substitutions Q222L, G224S, buffered saline (PBS) was delivered dropwise to stitution removes a potential N-linked glycosyl- and E627K that had been introduced by reverse the nostrils of the ferrets to induce sneezing. Ap- ation site (Asn-X-Thr/Ser; X, any amino acid) in genetics. Surprisingly, only two additional amino proximately 200 ml of the “sneeze” was collected HA and was detected in 99.6% of the A/H5N1wildtype acid substitutions, both in HA, were consistently in a Petri dish, and PBS was added to a final vol- sequences after 10 passages. T156Awas detected detected in all six airborne-transmissible viruses: ume of 2 ml. The nasal-wash samples were used in 89% of the A/H5N1HA Q222L,G224S PB2 E627K (i) H103Y (H, His; Y, Tyr), which forms part of for intranasal inoculation of the ferrets for the sequences after 10 passages, and the other 11% the HA trimer interface, and (ii) T156A, which is subsequent passages 7 through 10. We changed of sequences possessed the substitution N154K, proximal but not immediately adjacent to the the source of inoculum during the course of the which removes the same potential N-linked gly- RBS (fig. S8). Although we observed several experiment, because passaging nasal washes cosylation site in HA. other mutations, their occurrence was not con- may facilitate the selection of viruses that were In experiment 4 (see supplementary materials), sistent among the airborne viruses, indicating secreted from the URT. Because influenza viruses we investigated whether airborne-transmissible that of the heterogeneous virus populations gen- mutate rapidly, we anticipated that 10 passages viruses were present in the heterogeneous virus erated by passaging in ferrets, viruses with dif- would be sufficient for the virus to adapt to efficient population generated during virus passaging in ferent genotypes were transmissible. In addition, replication in mammals. ferrets (fig. S4). Nasal-wash samples, collected a single transmission experiment is not sufficient Virus titers in the nasal turbinates of ferrets at 3 dpi from ferrets at passage 10, were used to select for clonal airborne-transmissible viruses inoculated with A/H5N1wildtype ranged from ~1 × in transmission experiments to test whether because, for example, the consensus sequence 10 to 1 × 107 TCID50/gram tissue throughout 10 5 airborne-transmissible virus was present in the of virus isolated from F6 differed from the se- serial passages (Fig. 3A and fig. S7). In ferrets virus quasi-species. For this purpose, nasal-wash quence of parental virus isolated from F2. inoculated with A/H5N1HA Q222L,G224S PB2 E627K samples were diluted 1:2 in PBS and subsequent- Together, these results suggest that as few as virus, a moderate increase in virus titers in the ly used to inoculate six naïve ferrets intranasally: five amino acid substitutions (four in HA and one nasal turbinates was observed as the passage two for passage 10 A/H5N1wildtype and four for in PB2) may be sufficient to confer airborne trans- number increased. These titers ranged from 1 × passage 10 A/H5N1HA Q222L,G224S PB2 E627K virus. mission of HPAI A/H5N1 virus between mam- 104 TCID50/gram tissue at the start of the exper- The following day, a naïve recipient ferret mals. The airborne-transmissible virus isolate iment to 3.2 × 105 to 1 × 106 TCID50/gram tissue was placed in a cage adjacent to each inoculated with the least number of amino acid substitutions, in the final passages (Fig. 3A and fig. S7). No- donor ferret. These cages are designed to prevent compared with the A/H5N1wildtype, was recov- tably, virus titers in the nose swabs of animals direct contact between animals but allow airflow ered from ferret F5. This virus isolate had a total inoculated with A/H5N1HA Q222L,G224S PB2 E627K from a donor ferret to a neighboring recipient of nine amino acid substitutions; in addition to also increased during the successive passages, ferret (fig. S1) (27). Although mutations had ac- the three mutations that we introduced (Q222L with peak virus shedding of 1 × 105 TCID50 at cumulated in the viral genome after passaging and G224S in HA and E627K in PB2), this virus 2 dpi after 10 passages (Fig. 3B).These data in- of A/H5N1wildtype in ferrets, we did not detect harbored H103Y and T156A in HA, H99Y and dicate that A/H5N1HA Q222L,G224S PB2 E627K was replicating virus upon inoculation of MDCK I368V (I, Ile; V, Val) in PB1, and R99K (R, Arg) developing greater capacity to replicate in the cells with swabs collected from naïve recipient and S345N in NP (table S3). Reverse genetics ferret URT after repeated passage, with evidence ferrets after they were paired with donor ferrets will be needed to identify which of the five to nine for such adaptation becoming apparent by pas- inoculated with passage 10 A/H5N1wildtype virus amino acid substitutions in this virus are essential sage number 4. In contrast, virus titers in the nose (Fig. 5, A and B). In contrast, we did detect virus to confer airborne transmission. swabs of the ferrets collected at 1 to 4 dpi through- in recipient ferrets paired with those inoculated During the course of the transmission exper- out 10 serial passages with A/H5N1wildtype re- with passage 10 A/H5N1HA Q222L,G224S PB2 E627K iments with the airborne-transmissible viruses, vealed no changes in patterns of virus shedding. virus. Three (F1 to F3) out of four (F1 to F4) ferrets displayed lethargy, loss of appetite, and Passaging of influenza viruses in ferrets naïve recipient ferrets became infected as con- ruffled fur after intranasal inoculation. One of should result in the natural selection of hetero- firmed by the presence of replicating virus in eight inoculated animals died upon intranasal geneous mixtures of viruses in each animal with the collected nasal and throat swabs (Fig. 5, C inoculation (Table 1). In previously published a variety of mutations: so-called viral quasi- and D). A throat-swab sample obtained from experiments, ferrets inoculated intranasally with species (38). The genetic composition of the recipient ferret F2, which contained the highest WTA/Indonesia/5/2005 virus at a dose of 1 × 106 viral quasi-species present in the nasal washes virus titer among the ferrets in the first transmis- TCID50 showed neurological disease and/or www.sciencemag.org SCIENCE VOL 336 22 JUNE 2012 1539

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APPENDIX C 99 H5N1 death (39, 40). It should be noted that inoculation volunteers more than 70 years of age. The in- H103Y or T156A and E627K, have already been of immunologically naïve ferrets with a dose troduction of receptor-binding site mutations reported in public sequence databases (53); thus, of 1 × 106 TCID50 of A/H5N1 virus and the Q222L/G224S and the mutations H103Y and we conclude that these mutations do not appear subsequent course of disease is not representative T156A in HA, acquired during ferret passage, to have a detrimental effect on virus fitness. of the natural situation in humans. Importantly, did not result in increased cross-reactivity with Substitution H103Y has only been found once, although the six ferrets that became infected via human antisera (table S6), indicating that hu- in combination with T156A in a duck in China respiratory droplets or aerosol also displayed leth- mans do not have antibodies against the HA of (53). Substitution E627K in PB2 has been found argy, loss of appetite, and ruffled fur, none of the airborne-transmissible A/H5N1 virus that was in ~27% of avian A/H5N1 virus sequences and in these animals died within the course of the ex- selected in our experiments. ~29% of human A/H5N1 viruses (53). Substitu- periment. Moreover, previous infections of hu- Substitutions Q222L and G224S have pre- tion T156A in HA has been reported in >50% of mans with seasonal influenza viruses are likely to viously been shown to be sufficient to switch the viruses sequenced and was detected in 100% induce heterosubtypic immunity that would offer receptor-binding specificity of avian influenza of the viruses from human cases in Egypt (53). some protection against the development of se- strains (i.e., a-2,3–linked SA) to that of human Investigations of viral quasi-species during a vere disease (41, 42). It has been shown that mice strains (i.e., a-2,6–linked SA) (20, 35, 46, 47). massive avian influenza A/H7N7 virus outbreak and ferrets previously infected with an A/H3N2 Amino acid position 103 is distal from the RBS, in the Netherlands indicated that viruses with hu- virus are clinically protected against intranasal forms part of the trimer interface, and is unlikely man adaptation markers, including HA mutations challenge infection with an A/H5N1 virus (43, 44). to affect receptor specificity (fig. S8). T156 is that alter receptor specificity and mutations in After intratracheal inoculation (experiment 5; part of a N-glycosylation sequon, and T156A (as polymerase proteins that increase polymerase fig. S9), six ferrets inoculated with 1 × 106 TCID50 well as N154K) would delete this potential gly- activity like E627K in PB2, emerged rapidly in of airborne-transmissible virus F5 in a 3-ml vol- cosylation site (fig. S8); amino acid T156 is prox- poultry (55–57). Given the large numbers of ume of PBS died or were moribund at day 3. imal but not immediately adjacent to the RBS. HPAI A/H5N1 virus-infected hosts globally, the Intratracheal inoculations at such high doses do Loss of N-glycosylation sites at the tip of HA has high viral mutation rate, and the apparent lack of not represent the natural route of infection and are been shown to affect receptor binding of A/H1 detrimental effects on fitness of the mutations generally used only to test the ability of viruses to (48, 49) and the virulence of A/H5 virus (50). We that confer airborne transmission, it may simply cause pneumonia (45), as is done for vaccination- evaluated the impact of the HA mutations that be a matter of chance and time before a human- challenge studies. At necropsy, the six ferrets emerged during passaging in ferrets in a modified to-human transmissible A/H5N1 virus emerges. revealed macroscopic lesions affecting 80 to turkey red blood cell (TRBC) assay (Table 2). In The specific mutations we identified in these 100% of the lung parenchyma with average virus this assay, the binding of influenza viruses, with experiments that are associated with airborne titers of 7.9 × 106 TCID50/gram lung (fig. S10). a mutated HA, to normal TRBCs (expressing transmission represent biological traits that may These data are similar to those described previously both a-2,3–linked SA and a-2,6–linked SA) and be determined by a set of different amino acid for A/H5N1wildtype in ferrets (Table 1). Thus, al- modified TRBCs with either a-2,3–linked SA substitutions. For example, amino acid substitu- though the airborne-transmissible virus is lethal to or a-2,6–linked SA on the cell surface was eval- tions D701N (D, Asp) or S590G/R591Q in PB2 ferrets upon intratracheal inoculation at high doses, uated and compared to two reference viruses yield a similar phenotype to E627K (29). N182K the virus was not lethal after airborne transmission. with known receptor binding preference: avian and other substitutions in the RBS of HA may To test the effect of the mutations in HA in the A/H5N1 and human A/H3N2 viruses. As expected yield a similar phenotype to Q222L/G224S (35). airborne-transmissible virus on its sensitivity to and shown before, introduction of the Q222L and Such mutations should be considered for A/H5N1 antiviral drugs, we used virus isolated from F5 G224S mutations in the HA of A/H5N1 changed surveillance studies in outbreak areas. Imai et al. (experiment 6). This airborne-transmissible virus the receptor binding preference from a-2,3– recently identified different RBS changes (N220K, with nine amino acid substitutions displayed a linked SA to a-2,6–linked SA (35). Furthermore, Q222L) along with N154D (affecting the same sensitivity to the antiviral drug oseltamivir similar in our hands, the introduction of substitutions N-glycosylation sequon as T156A) and T314I in to that of A/H5N1wildtype (table S4). H103Yand T156A not only enhanced binding of HA as determinants of airborne transmission of In experiment 7, we evaluated the recognition A/H5N1HA Q222L,G224S PB2 E627K to a-2,6–linked an A/H5 virus (58). This airborne virus contained of the airborne-transmissible virus by antisera SA, as expected from glycan array studies (51), seven genes of the 2009 pandemic A/H1N1 vi- raised against potential A/H5N1 vaccine strains. but also increased the affinity for a-2,3–linked rus (which has S590G/R591Q in PB2 rather than Because only HA recognition by antibodies is SA. When these two mutations were introduced E627K), with the HA of A/H5N1 virus A/Vietnam/ evaluated in this assay, chimeric viruses were in the A/H5N1wildtype HA, the affinity for a-2,3– 1203/2004 (58). These data indicate that differ- generated based on six gene segments of the linked SA also increased. ent lineages of A/H5N1 virus and different amino mouse-adapted A/Puerto Rico/8/34 (PR8) virus Substitutions Q222L and G224S have previ- acid substitutions that affect particular biological with the HA and PB2 genes of the transmissible ously emerged in avian A/H2 and A/H3 viruses traits (receptor binding, glycosylation, replication) virus harboring amino acid substitutions H103Y, in nature (36, 52), and mutations associated with can yield airborne-transmissible A/H5N1 viruses. T156A, Q222L, and G224S in HA and E627K similar changes in receptor binding specificity Although our experiments showed that A/H5N1 in PB2. We replaced the MBCS of the HA by a have been detected repeatedly in A/H5 viruses— virus can acquire a capacity for airborne trans- monobasic cleavage site, allowing us to do these for instance, substitution N182K has been re- mission, the efficiency of this mode remains un- experiments under BSL2 conditions. The chimeric ported nine times (37, 51), which is why we clear. Previous data have indicated that the 2009 PR8/H5 virus reacted well with ferret antisera initially selected it for our investigations. The pandemic A/H1N1 virus transmits efficiently raised against A/Indonesia/5/2005 and several oth- other three substitutions we found consistently in among ferrets and that naïve animals shed high er prepandemic vaccine strains (table S5). In fact, airborne-transmissible viruses have all previously amounts of virus as early as 1 or 2 days after the presence of the four HA mutations increased been detected in HPAI A/H5N1 viruses circulat- exposure (27). When we compare the A/H5N1 the reactivity with H5 antisera by twofold or more. ing in the field (53). Only a minor fraction of the transmission data with that of reference (27), We subsequently used the same PR8/H5 A/H5N1 viruses that have circulated in outbreaks keeping in mind that our experimental design for chimeric virus in experiment 8 to evaluate the have been sequenced (estimated to be <0.001%) studying transmission is not quantitative, the data presence of existing immunity against the airborne- (53, 54). Yet the individual substitutions we shown in Figs. 5 and 6 suggest that A/H5N1 transmissible virus in sera obtained from human obtained, as well as combinations of T156A and airborne transmission was less robust, with less 1540 22 JUNE 2012 VOL 336 SCIENCE www.sciencemag.org

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