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Committee on Science, Technology, and Law; Policy and Global Affairs; Board on Life Sciences; Division on Earth and Life Studies; Forum on Microbial Threats; Board on Global Health; National Research Council; Institute of Medicine. Perspectives on Research with H5N1 Avian Influenza: Scientific Inquiry, Communication, Controversy: Summary of a Workshop. Washington (DC): National Academies Press (US); 2013 Apr 4.

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Perspectives on Research with H5N1 Avian Influenza: Scientific Inquiry, Communication, Controversy: Summary of a Workshop.

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Appendix CThe 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.

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

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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 infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors1-3. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus—comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus—that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.

Although H5N1 viruses continue to cause outbreaks in poultry and there are cases of human infection in Indonesia, Vietnam, Egypt and elsewhere (http://www.who.mt/mfluenza/human_animd_mterface/H5N1_cumulative_table_archives/en/index.html), they have not acquired the ability to cause human-to-human transmission. Investment in H5N1 vaccines has therefore been questioned. However, because humans lack immunity to influenza viruses possessing an H5 HA, the emergence of a transmissible H5-HA-possessing virus would probably cause a pandemic. To prepare better for such a scenario, it is critical that we understand the molecular changes that may render H5-HA-possessing viruses transmissible in mammals. Such knowledge would allow us to monitor circulating or newly emerging variants for their pandemic potential, focus eradication efforts on viruses that already have acquired subsets of molecular changes critical for transmission in mammals, stockpile antiviral compounds in regions where such viruses circulate, and initiate vaccine generation and large-scale production before a pandemic. Therefore, we studied the molecular features that would render H5-HA-possessing viruses transmissible in mammals.

Previous studies suggested that HA has a major role in host-range restriction of influenza A viruses1-3. The HA of human isolates preferentially recognizes sialic acid linked to galactose by α2,6-linkages (Siaα2,6Gal), whereas the HA of avian isolates preferentially recognizes sialic acid linked to galactose by α2,3–linkages (Siaα2,3Gal)3. A small number of avian H5N1 viruses isolated from humans show limited binding to human-type receptors, a property conferred by several amino acid changes in HA4-9. None of the H5N1 viruses tested transmitted efficiently in a ferret model10-13, although, while our paper was under review, one study14 reported that a virus with a mutant H5 HA and a neuraminidase (NA) of a human virus in the H5N1 virus background caused respiratory droplet transmission in one of two contact ferrets.

To identify novel mutations in avian H5 HAs that confer human-type receptor-binding preference, we introduced random mutations into the globular head (amino acids 120-259 (H3 numbering), which includes the receptor-binding pocket) of A/Vietnam/1203/2004 (H5N1; VN1203) HA (Supplementary Fig. 1). Although this virus was isolated from a human, its HA retains avian-type receptor-binding properties6,15. We also replaced the multibasic HA cleavage sequence with a non-virulent-type cleavage sequence, allowing us to perform studies in biosafety level 2 containment (http://www.who.int/csr/resources/publications/influenza/influenzaRMD2003_5.pdf). The mutated polymerase chain reaction (PCR) products were cloned into RNA polymerase I plasmids16 containing the VN1203 HA complementary DNA, which resulted in Escherichia coli libraries representing the randomly generated HA variants. Sequence analysis of 48 randomly selected clones indicated an average of 1.0 amino acid changes per HA globular head (data not shown). To generate an H5N1 virus library, plasmids for the synthesis of the mutated HA gene and the unmodified NA gene of VN1203 were transfected into human embryonic kidney (293T) cells together with plasmids for the synthesis of the six remaining viral genes of A/Puerto Rico/8/34 (H1N1; PR8), a laboratory-adapted human influenza A virus.

Figure 1. | Localization of amino acid changes identified in this study on the three-dimensional structure of the monomer of VN1203 HA (Protein Data Bank accession 2FK0)15.

Figure 1

| Localization of amino acid changes identified in this study on the three-dimensional structure of the monomer of VN1203 HA (Protein Data Bank accession 2FK0)15. a, Close-up view of the globular head of VN1203 HA. Mutations known to increase affinity (more...)

Turkey red blood cells (TRBCs; which possess both Siaα2,6Gal and Siaα2,3Gal on their surface (data not shown)) were treated with Salmonella enterica serovar Typhimurium LT2 sialidase, which preferentially removes α2,3–linked sialic acid (that is, avian-type receptors), creating TRBCs that predominantly possess Siaα2,6Gal on the cell surface (Siaα2,6-TRBCs; Supplementary Fig. 2). The virus library was then adsorbed to Siaα2,6-TRBCs at 4°C and extensively washed to remove nonspecifically or weakly bound viruses. Bound viruses were eluted by incubation at 37°C for 30 min, and then diluted to approximately ~0.5 viruses per well (on the basis of a pilot experiment that assessed the approximate number of eluted viruses). We screened one-third of the library (that is, 2.1 X 106viruses) in three separate selection experiments (that is, 0.7 X 106 viruses per experiment) and isolated 370 viruses that bound to Siaα2,6-TRBCs (Supplementary Fig. 2). Individual viruses were then grown in Madin-Darby canine kidney (MDCK) cells modified to overexpress Siaα2,6Gal (AX4 cells17), and screened again for their ability to agglutinate Siaα2,6-TRBCs (Supplementary Fig. 2). The parental control virus (designated VN1203/PR8) with avian-type receptor-binding specificity agglutinated untreated TRBCs (which possess both human-and avian-type receptors on their surface), but not TRBCs possessing predominantly human-type receptors (Siaα2,6-TRBCs; Supplementary Table 1). By contrast, of the 370 viruses originally isolated, nine agglutinated Siaα2,6-TRBCs, albeit with different efficiencies (Supplementary Table 1). All nine viruses possessed mutations in the region targeted for random mutagenesis; one mutant also possessed an additional mutation (E119G) in an area that was not targeted for mutation. Most of the mutations clustered around the receptor-binding pocket (Fig. 1a). Several of the selected viruses possessed mutations known to increase binding to human-type receptors, including N186K (ref. 9), S227N (ref. 5) and Q226L (which confers human-type receptor binding together with G228S)15 (all shown in blue in Fig. 1a). The identification of known determinants of human-type receptor-binding specificity from a library of random mutants validates our approach. Notably, our screen also identified mutations not previously associated with receptor-binding specificity.

Figure 2. | Characterization of the receptor-binding properties of isolated viruses.

Figure 2

| Characterization of the receptor-binding properties of isolated viruses. a, Binding of VN1203 mutants to sialylglycopolymers in solid-phase binding assays. A human virus (K173/PR8), an avian virus (VN1203/PR8) and mutant VN1203/PR8 viruses were compared (more...)

Table 1. |Transmission in ferrets inoculated with H5 avian-human reassortant viruses.

Table 1

|Transmission in ferrets inoculated with H5 avian-human reassortant viruses.

Although viruses were diluted to ~0.5 viruses per well for amplification in AX4 cells, we cannot exclude the possibility that some wells were infected with more than one virus, resulting in mixed populations. To confirm the significance of the identified mutations in HA for human-type receptor binding, the mutations were engineered into a VN1203/PR8 virus (possessing an avirulent HA cleavage site sequence, as described earlier). All nine mutants were generated; however, after two passages in MDCK cells, the S136N mutation reverted to the wild-type sequence. This mutant was excluded from further evaluation.

First, we confirmed the binding of the remaining eight variants to Siaα2,6-TRBCs (Supplementary Table 1). For comparison, we included a VN1203/PR8 virus with two changes in its HA (Q226L and G228S) previously shown to have increased binding to Siaα2,6Gal6,15. Indeed, compared to the wild-type VN1203/PR8 virus, the Q226L/G228S mutant displayed an increased ability to bind to human-type receptors. For the recreated variants, haemagglutination titres were higher and slightly different from the initial characterization, which we attribute to biological differences (the initial characterization was carried out with non-concentrated cell culture supernatant and potentially mixed virus populations, whereas the recreated viruses were concentrated and purified) and to experimental differences (that is, differences between the TRBC batches or the efficiency of α2,3-sialidase treatment, or both). Collectively, however, these experiments demonstrate that this random mutagenesis approach allows the identification of hitherto unrecognized amino acid substitutions that permit avian virus HAs to bind to human-type receptors.

To characterize further the receptor-binding properties of the selected variants, we used solid-phase binding assays in which sialylglycopolymers were absorbed to plates, which were then incubated with virus (Fig. 2a). A virus possessing the HA and NA genes of the seasonal human A/Kawasaki/173/2001 (H1N1; K173) virus and the remaining genes from PR8 (K173/PR8) served as a control virus with typical human-type receptor specificity. Indeed, K173/PR8 preferentially bound to Siaα2,6Gal. In contrast, VN1203/PR8 bound to only Siaα2,3Gal. As reported elsewhere6,15, the Q226L/G228S mutations led to increased binding to Siaα2,6Gal. Variants I202T/R220S, W153R/T160I, N169I/H184L/I217M and H130Q/K157E resembled VN1203/PR8 in their binding to glycans, despite the fact that these mutants weakly agglutinated Siaα2,6-TRBCs (see Supplementary Table 1). These viruses may have bound to glycans on TRBCs that were different from Siaα2,6Galβ1,4GlcNAc used in this study. However, variants N186K/M230I, S227N/G228A and Q226L/E231G showed an appreciable increase in binding to Siaα2,6Gal but also retained binding capacity for Siaα2,3Gal. Of all of the variants tested, only E119G/V152I/N224K/Q226L exhibited specificity for only Siaα2,6Gal. Thus, only one H5 HA variant with receptor-binding capability akin to that of seasonal influenza viruses was isolated from the library screen of 2.1 X 106 viruses. To identify the amino acid change(s) responsible for the conversion from Siaα2,3Gal to Siaα2,6Gal recognition in the E119G/V152I/N224K/Q226L virus HA, we tested the amino acid changes at positions 119, 152, 224 and 226 individually and in various combinations. Solid-phase binding assays demonstrated that the N224K/Q226L combination is critical for the shift from Siaα2,3Gal to Siaα2,6Gal recognition (Fig. 2b); Q226L in combination with V152I also conferred weak binding to α2,6-glycans.

To assess the effect of enhanced α2,6-glycan recognition on the attachment of viruses to human respiratory tracts, sections of tracheal and lung tissues were exposed to K173/PR8 (human-type receptor binder), VN1203/PR8 (avian-type receptor binder) and mutant VN1203/PR8 viruses (Fig. 2c). Because the N186K/M230I, S227N/G228A, Q226L/E231G, E119G/V152I/N224K/Q226L and N224K/Q226L mutants exhibited appreciablebinding to Siaα2,6Gal (Fig. 2a, b), the attachment of these mutants was also tested. On tracheal sections, theK173/PR8 virus bound extensively to ciliated epithelial cells (Fig. 2c and Supplementary Fig. 3), whereas the VN1203/PR8 virus bound poorly. By contrast, on lung sections, both viruses bound extensively to the alveolar epithelial surface (both type I and II pneumocytes; Fig. 2c and Supplementary Fig. 4). The binding patterns of these viruses correlate with the distribution of Siaα2,3 Gal (that is, avian-type receptors; present in lung epithelia) and Siaα2,6Gal (that is, human-type receptors; present in both trachea and lung epithelia) on the tissues, as observed with lectin staining'18 (Supplementary Fig. 5). Like the human K173/PR8 virus, the E119G/V152I/N224K/Q226L and N224K/Q226L mutants exhibited strong binding to the ciliated epithelial cells of the trachea (Fig. 2c and Supplementary Fig. 3). By contrast, the N186K/M230I, S227N/G228A and Q226L/E231G mutants displayed little-to-no binding to tracheal epithelia (Fig. 2c), despite their binding to Siaα2,6Gal (Fig. 2a). A number of sialylated oligosaccharides with differing branching patterns and chain lengths are thought to be present on the cell surface19. We therefore speculate that the mutants can recognize a short glycan structure such as Siaα2,6Galβ1,4GlcNAc, but may not recognize longer, more complex glycan structures, which arepossibly required forbindingto human tracheal epithelium. On the other hand, all mutants bound to alveolar epithelial cells (both type I and II pneumocytes; Fig. 2c and Supplementary Fig. 4). When the tissue sections were pre-treated with Arthrobacter ureafaciens sialidase (which cleaves all non-reducing terminally branched and unbranched sialic acids), virus binding to the tissues was substantially reduced (Supplementary Fig. 6a-c), confirming the sialic acid binding specificity of the virus. These data indicate that alterations in the receptor specificity of the E119G/V152I/N224K/Q226L and N224K/Q226L mutants have profound effects on virus attachment to human respiratory epithelium.

Figure 3. | Virus replication in respiratory organs.

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 and 6 after infection for virus titration. Virus titres in nasal turbinates, trachea and lung were determined (more...)

Figure 4. | Respiratory droplet transmission of H5 avian-human reassortant viruses in ferrets.

Figure 4

| Respiratory droplet transmission of H5 avian-human reassortant viruses in ferrets. a-f, Groups of three, five, or six ferrets were inoculated intranasally with 106 p.f.u. of rgCA04 (a), rgVN1203/CA04 (b), rg(N224K/Q226L)/CA04 (c), HA(N158D/N224K/Q226L)/CA04 (more...)

Figure 5. | Polykaryon formation by HeLa cells expressing wild-type or mutant HAs after acidification at low pH.

Figure 5

| Polykaryon formation by HeLa cells expressing wild-type or mutant HAs after acidification at low pH. a, The efficiency of polykaryon formation over a pH range of 5.4-6.0 was estimated from the number of nuclei in polykaryons divided by the total number (more...)

Figure 6. | Effect of heat treatment on the infectivity and haemagglutination activity of viruses.

Figure 6

| Effect of heat treatment on the infectivity and haemagglutination activity of viruses. Aliquots of a virus stock containing 128 HA units were incubated for the times indicated at 50°C. a, Virus titres in heat-treated samples were determined (more...)

In an avian H3 HA, the Q226L mutation changed the binding preference from avian-to human-type20. A previous study found that the Q226L mutation on an H5 HA does not confer efficient binding to α2,6-glycans in a glycan array15; however, when tested in combination with G228S, increased binding to human-type receptors, but not a complete switch from avian-to human-type receptor-binding specificity, was observed15. By contrast, here we found that Q226L in combination with N224K resulted in a switch from Siaα2,3Gal to Siaα2,6Gal binding in an H5 HA and allowed virus binding to human 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 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 avian H5 HA is closer to the opposing 130-loop than in human H3 HA, indicating that a wider binding site for human H3 HA, compared to that of avian H5 HA, may be required to optimize contacts with the larger Siaα2,6-glycans21. N224 lies on the turn leading into the 220-loop, adjacent to position 226 (Fig. 1a). Replacement of N224 may alter the orientation of the 220-loop and thus optimize contacts between L226 and Siaα2,6Gal-containing receptors, thereby increasing the preference for α2,6 linkages.

Recent studies reported that 2009 pandemic H1N1 and H5N1 viruses show high genetic compatibility22,23. These two viruses have been isolated from pigs24-28, which have been considered as ‘mixing vessels’ for the reassortment of avian, swine and human strains. Thus, the coexistence of H5N1 and 2009 pandemic H1N1 viruses could provide an opportunity for the generation of transmissible H5 avian-human reassortants in mammals. Therefore, we generated reassortant viruses possessing the mutant VN1203 HAs generated above, and the seven remaining gene segments from a prototype 2009 pandemic H1N1 virus (A/California/04/2009, CA04). Experiments with viruses possessing the wild-type HA cleavage site were performed in enhanced biosafety level 3 (BSL3+) containment laboratories approved for such use by the Centers for Disease Control and Prevention (CDC) and the United States Department of Agriculture (USDA). Because efficient human-to-human transmission is a critical feature of pandemic influenza viruses, we examined the growth and transmissibility of reassortant viruses in ferrets, which are widely accepted as an animal model for influenza virus transmissibility and pathogenesis studies. Because the E119G/V152I/N224K/Q226L and N224K/Q226L variants bound extensively to human tracheal epithelia (Fig. 2c), we generated by reverse genetics (rg) three H5 reassortant viruses possessing the VN1203 HA or mutant HAs (all with the wild-type multibasic cleavage site) and the remaining genes from the CA04 virus. The VN1203 HA mutants tested included the one containing four mutations, E119G, V152I, N224K and Q226L (designated rg(E119G/V152I/N224K/Q226L)/CA04), and another containing two mutations, N224K and Q226L (designated rg(N224K/Q226L)/CA04).

To determine whether the introduced HA mutations affected the replication of the H5 reassortant viruses, six ferrets were inoculated intranasally with 106 plaque-forming units (p.f.u.) of virus. On day 3 after infection, a recombinant virus whose genes all came from CA04, rgCA04, replicated efficiently in the respiratory organs of infected animals, and was isolated from the colon, but not from any other organs tested (Fig. 3 and Supplementary Table 2). A virus possessing H5 VN1203 HA and the remaining genes from CA04 (designated rgVN1203/CA04) replicated to titres comparable to those of rgCA04 in nasal turbinates, but substantially less in the lungs. By contrast, the two H5 reassortant viruses with HA mutations (rg(E119G/V152I/N224K/Q226L)/CA04 and rg(N224K/Q226L)/CA04) were severely limited in their replicative ability in trachea. Although virus titres in nasal turbinates and lung were not statistically different between rg(N224K/Q226L)/CA04 and rgCA04, the virus titre in nasal turbinates was significantly lower in animals inoculated with rg(E119G/V152I/N224K/Q226L)/CA04 than in animals inoculated with rgCA04 (Dunnett's test; P = 0.0002; Fig. 3). Notably, rgVN1203/CA04 (avian-type receptor binder) replicated efficiently in nasal turbinates of ferrets, which have a similar sialic acid receptor distribution pattern to that of the human respiratory tract29,30. The reason for this discrepancy is unclear; however, replication of avian H5N1 viruses in ferret nasal turbinates has been reported12,13.

Table 2. Receptor specificity of the different mutant A/H5N1 viruses, as determined by a modified TRBC hemagglutination assay.

Table 2

Receptor specificity of the different mutant A/H5N1 viruses, as determined by a modified TRBC hemagglutination assay.

Although virus titres in respiratory organs were generally lower on day 6 after infection than on day 3 after infection, rg(N224K/Q226L)/CA04 still showed high levels of replication at day 6 after infection; titres in nasal turbinates ranged from 104.6 to 108.1 p.f.u. g–1 (Fig. 3). Sequence analysis of viruses in nasal turbinates on day 6 after infection revealed that viruses in ferret 2 and ferret 3 possessed N158D and N158K mutations in their HA (in addition to the original two mutations), respectively, leading to the loss of the glycosylation site at position 158 (that is, 158N-S-T to 158D-S-T or 158K-S-T; Fig. 1a and Supplementary Table 3). In nasal turbinates on day 6 after infection, the titre of the virus with the N158D/N224K/Q226L mutations (108.1 p.f.u.g–1; see Fig. 3, ferret 2 of rg(N224K/Q226L)/CA04) was approximately four orders of magnitude higher than that of the original rg(N224K/Q226L)/CA04 (104.6p.f.u. g–1; Fig. 3, ferret 1 of rg(N224K/Q226L)/CA04), whereas the virus with the N158K/N224K/Q226L mutations (105.6p.f.u.g–1; Fig. 3, ferret 3 of rg(N224K/Q226L)/CA04) grew to one order of magnitude higher than the original mutant. These data indicate that the additional mutation N158D improved the replication of rg (N224K/Q226L)/CA04 in ferrets. To test the effect of this mutation on the replication of H5 reassortant viruses in ferrets, we examined the replicative ability of a virus with the triple N158D/N224K/Q226L HA substitutions in ferrets. This HA(N158D/N224K/Q226L)/CA04 virus replicated efficiently in infected animals, except in the trachea (Fig. 3 and Supplementary Table 2). On day 3 after infection, this virus was isolated from the brain of two of the three animals tested, although we did not observe neurological signs in these animals. These results indicate that the N158D mutation contributed to the efficient growth in the nasal turbinates of ferrets of an H5 reassortant virus with the N224K/Q226L mutations. Removal of the glycosylation site at position 158 has been reported to result in enhanced binding of H5N1 viruses to human-type receptors in combination with the Q226L/G228S mutations7. A previous study showed that H5N1 viruses lacking this glycosylation site transmit efficiently by direct contact among guinea-pigs31. By contrast, H5N1 viruses that acquire this glycosylation site lose the ability to transmit among guinea-pigs. Therefore, we speculated that the loss of the glycosylation site in HA(N158D/N224K/Q226L)/CA04 virus may affect its transmissibility in ferrets.

To assess the ability of H5 reassortant viruses with human-type receptor specificity to transmit between ferrets, we placed naive ferrets in wireframe cages next to ferrets inoculated with 106 p.f.u. of rgCA04, rgVN1203/CA04, rg(N224K/Q226L)/CA04, or HA(N158D/N224K/Q226L)/CA04 (Supplementary Fig. 7). Similar to previous experiments32, rgCA04 was efficiently transmitted via respiratory droplets to all three contact ferrets, as evidenced by the detection of virus in nasal washes and haemagglutination inhibition (HI) antibody in these animals (Table 1 and Fig. 4). By contrast, rgVN1203/CA04 and rg(N224K/Q226L)/CA04 were not transmitted; neither virus shedding nor seroconversion was detected in any contact animals, despite the binding of the latter to Siaα2,6Gal. This result was consistent with that of previous studies in which human-type receptor recognition was shown to be necessary but not sufficient for respiratory droplet transmission of an H5N1 virus in a ferret model12,14. In the HA(N158D/N224K/Q226L)/CA04-inoculated group, virus was recovered from two of the six contact ferrets (pairs 1 and 2) between days 5 and 7 after contact. Moreover, seroconversion was detected in five animals including those from which virus was recovered. No animals died in the course of these transmission experiments. This finding demonstrates the generation of an H5 HA that supports virus transmission by respiratory droplets among ferrets.

Figure 7. | Pathological analyses of H5 avian-human reassortant viruses.

Figure 7

| Pathological analyses of H5 avian-human reassortant viruses. a, Representative histological changes in nasal turbinates from influenza-virus-infected ferrets. Three ferrets per group were infected intranasally with 106p.f.u. of virus, and tissues were (more...)

To determine whether additional mutations occurred in the HA of HA(N158D/N224K/Q226L)/CA04 during transmission, viral RNA was analysed from nasal washes of inoculated and contact ferrets (Fig. 4 and Supplementary Table 4). On day 5 after infection, the A242S andT318I mutations in HA were present in five (pairs 1,3,4,5 and 6) and one (pair 2) of the six inoculated animals, respectively. Viruses derived from the contact animals of pair 1 on day 7 after contact had two changes in HA (K193N and A242S) (Fig. 1a), whereas those derived from the contact animals of pair 2 contained a single change in HA (T318I) (Fig. 1b), indicating that additional changes in HA occurred during the infection of ferrets with HA(N158D/N224K/Q226L)/CA04. No mutations in the remaining genes were detected in any of these viruses from nasal washes compared with the CA04 virus sequences.

Because HA(N158D/N224K/Q226L)/CA04 was isolated from only one-third of the contact animals, we isolated a virus from the nasal wash of the contact ferret that shed a high titre (107.5 p.f.u. ml–1 ) of virus on day 7 after contact (pair 2) (Fig. 4d) to evaluate the replication and transmissibility of that virus in ferrets. This mutant virus, designated HA(N158D/N224K/Q226L/T318I)/CA04, replicated efficiently in the nasal turbinates and was isolated from brain tissue (Fig. 3 and Supplementary Table 2). In the transmission study, four of the six contact ferrets were positive for virus between days 3 and 7 after contact, and all contact animals were seropositive; no animals died in the course of the transmission experiments (Table 1; Fig. 4e and Supplementary Fig. 8). Notably, this transmission pattern is comparable to that of the 1918 pandemic H1N1 virus when tested under the same experimental conditions; the 1918 pandemic virus was recovered from the nasal wash of two of three contact animals (our own unpublished data). Sequence comparison of viruses from inoculated and contact animals identified mutations at positions 225 and 242 as well as a reversion at position 224 (Fig. 1a and Supplementary Table 5) (in addition to the original four mutations) although the 224 reversion was found only in viruses from inoculated ferrets. Collectively, these findings demonstrate that four amino acid substitutions (N158D/N224K/Q226L/T318I) in H5 HA confer efficient respiratory droplet transmission in ferrets to a virus possessing an H5 HA in a 2009 pandemic H1N1 backbone. We also confirmed that recombinant viruses possessing the three HA mutations N158D, N224K and Q226L, or the four HA mutations N158D, N224K, Q226L and T318I, and the NA of VN1203 in a PR8 backgrand (designated N158D/N224K/Q226L or N158D/N224K/Q226L/T318I, respectively) preferentially bind to Siaα2,6Gal and attach to human tracheal epithelia (Fig. 2c, d).

HA(N158D/N224K/Q226L/T318I)/CA04 transmitted by respiratory droplet more efficiently than HA(N158D/N224K/Q226L)/CA04, raising the possibility that the T318I mutation is involved in the efficient transmission of avian H5N1/pandemic H1N1 reassortants. To explore the functional role of this mutation in respiratory droplet transmission, we generated an H5 reassortant expressing the H5 HA with the T318I mutation and examined its receptor-binding specificity and transmissibility. This reassortant (designated rgT318I/CA04) bound to only Siaα2,3Gal and showed little binding to human tracheal epithelia (Fig. 2c, d). rgT318I/CA04 did not transmit via respiratory droplet among ferrets (Table 1 and Fig. 4f), although it replicated in nasal turbinates and trachea as efficiently as rgCA04 (Fig. 3 and Supplementary Table 2). These results indicate that the T318I mutation alone is not sufficient for H5 reassortant viruses to transmit efficiently among ferrets.

Influenza virus HA protein has membrane-fusion as well as receptor-binding activity. Notably, in the three-dimensional model of influenza A virus HA, residue 318 is located proximally to the fusion peptide (Fig. 1b), which has key roles in the membrane fusion process. To assess the effect of HA mutations on low-pH-induced membrane fusion activity, we examined the pH at which the fusion activity of wild-type and mutant HA was activated (Fig. 5). The wild-type HA had a threshold for membrane fusion of pH 5.7; the N224K/Q226L and N158D/N224K/Q226L mutations raised the threshold for fusion to >pH5.9, whereas the T318I mutation reduced the threshold for fusion to pH 5.5. The N158D/N224K/Q226L/T318Imutations showed wild-type fusogenic properties (that is, a threshold at pH 5.7). The HA of influenza virus undergoes a low-pH-dependent conformational change, which is required for fusion of the viral envelope with the target membrane33. Such a conformational change to a fusion-active form can also lead to viral inactivation. Therefore, sustained and efficient human-to-human transmission of virus may require a certain level of stability of the HA protein in an acidic environment, as the pH of human nasal mucosa, where human influenza viruses replicate primarily, is approximately pH 5.5-6.5 (ref. 34). Our findings suggest that an increase in the pH threshold for fusion as a result of the N224K/Q226L mutations that shift the HA receptor recognition from avian-type to human-type may reduce HA protein stability; however, the T318I mutation decreases the pH threshold for fusion activity, resulting in a stable mutant HA.

Because heat treatment at neutral pH is also known to promote a fusogenic form of HA protein35,36 and serve as a surrogate assay for HA stability37, we next tested whether the HA mutations described above affect the heat stability of the HA protein. Wild-type and mutant HA viruses were incubated at 50°C for various times, after which the loss of infectivity and haemagglutination activity were determined. The wild-type and N224K/Q226L viruses lost most of their infectivity by heating for 60 min (>5.5-log10 decrease in titre; Fig. 6a), whereas the N158D/N224K/Q226L and N158D/N224K/Q226L/T318I mutants exhibited considerable tolerance to high temperature (3.9- and 3.4-log10 decrease after a 60-min incubation, respectively) and the T318I mutant was most resistant (only a 1.4-log10 decrease under the same conditions). In haemagglutination assays, the N224K/Q226L mutant HA lost activity more rapidly than did the wild-type HA, and N158D/N224K/Q226L lost activity more rapidly than did the N158D/N224K/Q226L/T318I mutant (Fig. 6b). Thus, addition of the N158D mutation to the N224K/Q226L HA increased HA stability and subsequent addition of the fourth mutation, T318I, rendered the HA protein even more stable. Taken together, these results suggest that the addition of the T318I mutation to H5 HAs that preferentially recognize human-type receptors restores HA protein stability, thereby allowing a virus carrying the N158D/N224K/Q226L/T318I mutations in HA to transmit efficiently via respiratory droplet among ferrets. In conclusion, a fine balance of mutations affecting different functions in HA (such as receptor-binding specificity and HA stability) may be critical to confer transmissibility in ferrets.

We next compared the pathogenicity in ferrets of H5 avian-human reassortants with that of the pandemic H1N1 virus CA04 (Fig. 7, Supplementary Information and Supplementary Figs 9-11). The control virus, rgCA04, caused substantial body weight loss (15.1%) (Table 1 and Supplementary Fig. 9). By contrast, the four reassortant viruses caused only modest weight loss (<10%) in most of the animals. However, no statistically significant differences in body weight loss were found between the reassortant viruses and rgCA04. Pathological examination revealed similar histological changes and levels of viral antigens in the nasal mucosa of rgCA04-, HA(N158D/N224K/Q226L)/CA04- and HA(N158D/N224K/Q226L/T318I)/CA04-infected ferrets (Fig. 7a, b). In the rgVN1203/CA04 and rg(N224K/Q226L)/CA04 groups, however, less tissue damage was found in the nasal mucosa compared with the rgCA04 group on day 3 after infection (Dunnett's test; P = 0.0057 and 0.0175, respectively; Fig. 7b). In addition, all three viruses caused lung lesions (Supplementary Information and Supplementary Figs 10 and 11).

To assess whether current control measures may be effective against the H5 transmissible reassortant mutant virus, we examined the reactivity of sera from individuals vaccinated with an H5N1 prototype vaccine38 against a virus possessing the N158D/N224K/Q226L/T318I mutations in HA. We found that pooled human sera from individuals immunized with this vaccine reacted with the virus possessing the mutant H5 HA (N158D/N224K/Q226L/T318I) at a higher titre than with a wild-type H5 HA virus (VN1203/PR8; Supplementary Table 6), indicating that current H5N1 vaccines would be efficacious against the H5 transmissible reassortant mutant virus. In addition, the H5 transmissible reassortant mutant virus (HA(N158D/N224K/Q226L/T318I)/CA04) was highly susceptible to a licensed NA inhibitor, oseltamivir (Supplementary Table 7). These experiments show that appropriate control measures would be available to combat the transmissible virus described in this study.

Currently, we do not know whether the mutations that we identified in this study that allowed the HA(N158D/N224K/Q226L/T318I)/CA04 virus to be transmissible in ferrets would also support sustained human-to-human transmission. In particular, we wish to emphasize that the transmissible HA(N158D/N224K/Q226L/T318I)/CA04 virus possesses seven segments (all but the HA segment) from a human pandemic 2009 H1N1 virus. Human-virus-characteristic amino acids in these seven segments may have critically contributed to the respiratory droplet transmission of the HA(N158D/N224K/Q226L/T318I)/CA04 virus in ferrets. Examples include amino acids in the PB2 polymerase protein that confer efficient replication in mammalian, but not avian, cells39-43. As the PB2 gene of the HA(N158D/N224K/Q226L/T318I)/CA04 virus is of human virus origin, the virus possesses high replicative ability in mammalian cells. In contrast, most avian virus PB2 proteins lack these human-type amino acids, although one of these changes (a glutamic-acid-to-lysine mutation at position 627) is found in highly pathogenic avian H5N1 viruses circulating in the Middle East44. As a second example, the viral NA gene may contribute to viral transmissibility. The NA protein cleaves a-ketosidic linkages between a terminal sialic acid and an adjacent sugar residue, an activity that balances the sialic-acid-binding activity of HA. A recent study found that a human virus NA gene was critical to confer limited transmissibility to a mutant H5 avian-human reassortant virus14. In general, a human-type receptor recognizing H5 HA alone may not be sufficient to confer transmissibility in mammals, but may have to act together with other human-virus-characteristic traits (in PB2, NA, and/or other viral proteins). Therefore, at this point we cannot predict whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible.

Three of the residues identified here (N224, Q226 and T318) have been strictly conserved among H5 HA proteins isolated since 2003. However, as H5N1 viruses continue to evolve and infect people, receptor-binding variants of H5N1 viruses, including avian-human reassortant viruses as tested here, may emerge. One of the four mutations we identified in our transmissible virus, the N158D mutation, results in loss of a glycosylation site. Many H5N1 viruses isolated in the Middle East, Africa, Asia and Europe do not have this glycosylation site. Therefore, only three nucleotide changes are needed for the HA of these viruses to support efficient transmission in ferrets. In addition, the H5N1 viruses circulating in these geographic areas also possess a glutamic-acid-to-lysine mutation at position 627 in the PB2 protein, which promotes viral replication in certain mammals, including humans40,45. Therefore, these viruses may be several steps closer to those capable of efficient transmission in humans and are of concern.

Our study highlights the pandemic potential of viruses possessing an H5 HA. Although current vaccines may protect against a virus similar to that tested here, the continued evolution of H5N1 viruses reinforces the need to prepare and update candidate vaccines to H5 viruses. The amino acid changes identified here will help individuals conducting surveillance in regions with circulating H5N1 viruses (for example, Egypt, Indonesia, Vietnam) to recognize key residues that predict the pandemic potential of isolates. Rapid responses in a potential pandemic situation are essential in order to generate appropriate vaccines and initiate other public health measures to control infection. Furthermore, our findings are of critical importance to those making public health and policy decisions.

Our research answers a fundamental question in influenza research: can H5-HA-possessing viruses support transmission in mammals? Moreover, our findings have suggested that different mechanisms (that is, receptor-binding specificity and HA stability) may act in concert for efficient transmissibility in mammals. This knowledge will facilitate the identification of additional mutations that affect viral transmissibility; the monitoring of this expanded set of changes in natural isolates may improve our ability to assess the pandemic potential of H5N1 viruses. Thus, although a pandemic H5N1 virus may not possess the amino acid changes identified in our study, the findings described here will advance our understanding of the mechanisms and evolutionary pathways that contribute to avian influenza virus transmission in mammals.

METHODS SUMMARY

Viruses

All recombinant viruses were generated by using reverse genetics essentially as described previously16. All experiments with the viruses possessing the wild-type HA cleavage site were performed in an enhanced biosafety level 3 (BSL3+) containment laboratory approved for such use by the CDC and the USDA.

Infection and transmission in ferrets

Six-ten-month-old female ferrets (Triple F Farms) were intramuscularly anaesthetized and intranasally inoculated with 106 p.f.u. (500 μl) of virus. On days 3 and 6 after infection, ferrets were killed for virological and pathological examinations. The virus titres in various organs were determined by use of plaque assays in MDCK cells.

For transmission studies in ferrets, animals were housed in adjacent transmission cages that prevented direct and indirect contact between animals but allowed spread of influenza virus through the air (Showa Science; Supplementary Fig. 7). Ferrets were intranasally inoculated with 106 p.f.u. (500 μl) of virus (inoculated ferrets). Twenty-four hours after infection, naïve ferrets were each placed in a cage adjacent to an inoculated ferret (contact ferrets). To assess viral replication in the nasal turbinates, we determined viral titres in nasal washes collected from virus-inoculated and contact ferrets on day 1 after inoculation or co-housing, respectively, and then every other day. Animal studies were performed in accordance with Animal Care and Use Committee guidelines of the University of Wisconsin-Madison.

Biosafety and biosecurity

All recombinant DNA protocols were approved by the University of Wisconsin-Madison's Institutional Biosafety Committee after risk assessments were conducted by the Office of Biological Safety, and by the University of Tokyo's Subcommittee on Living Modified Organisms, and, when required, by the competent minister of Japan. In addition, the University of Wisconsin-Madison Biosecurity Task Force regularly reviews the research program and ongoing activities of the laboratory. The task force has a diverse skill set and provides support in the areas of biosafety, facilities, compliance, security and health. Members of the Biosecurity Task Force are in frequent contact with the principal investigator and laboratory personnel to provide oversight and assure biosecurity. Experiments with viruses possessing the wild-type HA cleavage site were performed in enhanced BSL3 containment laboratories approved for such 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 minimum of 6 years of experience with highly pathogenic influenza viruses and animal studies with highly pathogenic viruses. Our staff wear powered air-purifying respirators that filter the air, and disposable coveralls; they shower out on exit from the facility. The containment facilities at University of Wisconsin-Madison were designed to exceed standards outlined in Biosafety in Microbiological and Biomedical Laboratories (5th edition; http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf). Features of the BSL3-enhanced suites include entry/exit through a shower change room, effluent decontamination, negative air-pressure laboratories, double-door autoclaves, double HEPA-filtered exhaust air, and gas decontamination ports. The BSL3-Agriculture suite features include all those listed for BSL3-enhanced plus HEPA-filtered supply and double-HEPA-filtered exhaust air, double-gasketed watertight and airtight seals, airtight dampers on all ductwork, and the structure was pressure-decay tested during commissioning. The University of Wisconsin-Madison facility has a dedicated alarm system that monitors all building controls and sends alarms (~500 possible alerts). Redundancies and emergency resources are built-in to the facility including two air handlers, two compressors, two filters each place filters are needed, two effluent sterilization tanks, two power feeds to the building, an emergency generator in case of a power failure and other physical containment measures in the facility that operate without power. Biosecurity monitoring of the facility is ongoing. All personnel undergo Select Agent security risk assessment by the United States Criminal Justice Information Services Division and complete rigorous biosafety, BSL3 and Select Agent training before participating in BSL3-level experiments. Refresher training is scheduled on a regular basis. The principal investigator participates in training sessions and emphasizes compliance to maintain safe operations and a responsible research environment. The laboratory occupational health plan is in compliance with the University of Wisconsin-Madison Occupational Health Program. Select agent virus inventory is checked monthly and submitted to the University of Wisconsin-Madison Research Compliance Specialist. Virus inventory is submitted 1-2 times per year to the file holder in the Select Agent branch of the CDC. The research program, procedures, occupational health plan, documentation, security and facilities are reviewed annually by the University of Wisconsin-Madison Responsible Official and at regular intervals by the CDC and the Animal and Plant Health Inspection Service (APHIS) as part of the University of Wisconsin-Madison Select Agent Program.

Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

Received 18 August 2011; accepted 9 March 2012. Published online 2 May 2012.

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Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Acknowledgements The authors would like to acknowledge D. Holtzman for his contributions to the initial concept for this project and thoughtful scientific discussions. We thank M. McGregor, R. Moritz, L. Burley, K. Moore, A. Luka, J. Bettridge, N. Fujimoto and M. Ito for technical support, S. Watson for editing the manuscript, and the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam for the A/Vietnam/1203/2004 (H5N1) virus, which was obtained from the CDC. This work was supported by the Bill & Melinda Gates Foundation (Grants 48339 and OPPGH5383), by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by ERATO (Japan Science and Technology Agency), and by the National Institute of Allergy and Infectious Diseases Public Health Service Research grants. The following reagents were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: polyclonal anti-monovalent influenza subvirion vaccine rgA/Vietnam/1203/2004 (H5N1), (antiserum, Human), high tire pool, NR-4109 and low titre pool, NR-4110.

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., 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., 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., 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. analysed the data; M.I., T.W., M.H., S.C.D., K.S., E.A.M., G.N. and Y.K. wrote the manuscript; M.I., T.W. and M.H. contributed equally to this work.

Author Information Reprints and permissions information is available at www.nature.com/reprints. This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence, and is freely available to all readers at www.nature.com/nature. The authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/nature. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Correspondence and requests for materials should be addressed to Y.K. (kawaokay@svm.vetmed.wisc.edu).

METHODS

Cells

Madin-Darby canine kidney (MDCK) cells and MDCK cells overexpressing Siaα2,6Gal (AX4 cells17) were maintained in Eagle's minimal essential medium (MEM) containing 5% newborn calf serum. Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). HeLa cells were maintained in MEM containing 10% FBS. All cells were maintained at 37°C in 5% CO2.

Plasmid construction and reverse genetics

Plasmid constructs for viral RNA production (pPolI)-containing the genes of the A/Vietnam/1203/2004 (H5N1; VN1203), A/Puerto Rico/8/34 (H1N1; PR8), A/Kawasaki/173/2001 (H1N1; K173) and A/California/04/2009 (H1N1; CA04) viruses flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—were constructed as described16. The multibasic amino acids at the haemagglutinin (HA) cleavage site (RERRRKKR↓.G) of the reassortant viruses between VN1203 and PR8 were changed to RETR↓G by site-directed mutagenesis. All transfectant viruses were generated by using reverse genetics essentially as described previously16. Recombinant viruses were amplified in MDCK or AX417 cells and stored at –80°C until use. The HA segment of all viruses was sequenced to ensure the absence of unwanted mutations. All experiments with the reassortant viruses between VN1203 and CA04 were performed in enhanced biosafety level 3 containment laboratories approved for such use by the CDC and the USDA.

To introduce random mutations into the globular head of the VN1203 HA protein, a 143-amino-acid region spanning residues 120-259 (H3 numbering) was selected. This region was subjected to PCR-based random mutagenesis by use of the GeneMorph II kit (Stratagene) following the manufacturer's instructions. The targeted mutation rate (1-2 amino acid replacements per molecule) was achieved through optimization of the template quantity, and was confirmed by sequence analysis of 48 individual clones. By using a PCR-based cloning strategy, we inserted the mutagenized region into its respective vector containing the VN1203 HA gene between the human RNA polymerase I promoter and mouse RNA polymerase I terminator sequences. The composition of the plasmid library was confirmed by sequencing. The plasmid library was then used to generate an influenza virus library, essentially as described16. The size of the virus library was 7 X 106p.f.u.

Preparation of sialidase-treated TRBCs

Turkey red blood cells (TRBCs) were washed three times with phosphate-buffered saline (PBS), and diluted to 20% (vol/vol) in PBS. TRBCs (1 ml) were incubated with 500 U of α2,3-sialidase from Salmonella enterica serovar Typhimurium LT2 (NEB) for 20-24 h at 37°C, washed three times in PBS, and re-suspended in PBS or MEM containing 1% bovine serum albumin (BSA) (MEM/BSA).

Haemagglutination assay

Viruses (50 μl) were serially diluted with 50 μl of PBS in a microtitre plate. An equal volume (that is, 50 μl) of a 0.5% (vol/vol) TRBC suspension was added to each well. The plates were kept at room temperature and haemagglutination was assessed after a 1-h incubation.

Virus library screening

To select VN1203 HA variants that had acquired the ability to recognize human-type receptors, three parallel experiments were carried out, each with 0.7 X 106 viruses. The virus library was first incubated with 0.1 ml of 10% (vol/vol) α2,3-sialidase-treated TRBCs for 10 min at 4°C. After this incubation, the TRBCs and bound viruses were pelleted at 1,000 r.p.m. for 1 min, and the pellets then washed ten times in MEM/BSA containing 313 mM NaCl. Bound viruses were eluted by incubation at 37°C for 30 min and then diluted to approximately 0.5 virus per well (determined by virus titration in a pilot study). Individual viruses were then amplified in AX4 cells, which overexpress Siaα2,6Gal17. Individual viruses were re-screened by using haemagglutination assays with α2,3-sialidase-treated TRBCs.

Solid-phase binding assay

Viruses were grown in MDCK cells, clarified by low-speed centrifugation, laid over a cushion of 30% sucrose in PBS, and ultracentri-fuged at 25,000 r.p.m. for 2 h at 4°C. Virus stocks were aliquoted and stored at –80°C. Virus concentrations were determined by using haemagglutination assays with 0.5% (vol/vol) TRBCs. The direct receptor-binding capacity of viruses was examined by use of a solid-phase binding assay as previously described9. Microtitre plates (Nunc) were incubated with the sodium salts of sialylglycopolymers (poly-L-glutamic acid backbones containing N-acetylneuraminic acid linked to galactose through either an α2,3 (Neu5Aca2,3Galβ1,4GlcNAcβ1-pAP) or an α2,6 (Neu5Acα2,6Galp1,4GlcNAcp1-pAP) bond) in PBS at 4°C overnight. After the glycopolymer solution was removed, the plates were blocked with 0.15 ml of PBS containing 4% BSA at room temperature for 1 h. After four successive washes with ice-cold PBS, the plates were incubated in a solution containing influenza virus (8-32 HA units in PBS) at 4°C overnight. After washing as described above, the plates were incubated for 2 h at 4°C with rabbit polyclonal antiserum to either K173 or VN1203 virus. The plates were then washed again as before and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antiserum for 2 h at 4°C. After washing, the plates were incubated with O-phenylenediamine (Sigma) in PBS containing 0.01% H2O2 for 10 min at room temperature, and the reaction was stopped with 0.05 ml of 1 M HCl. The optical density at 490 nm was determined in a plate reader (Infinite M1000; Tecan).

Virus binding to human airway tissues

Paraffin-embedded normal human trachea (US Biological) and lung (BioChain) tissue sections were deparaffinized and rehydrated. Sections were then blocked by using 4% BSA in PBS and covered with virus suspensions (64 HA units in PBS) at 4°C overnight. After being washed four times in ice-cold PBS, the sections were incubated with primary antibodies for 3 h at 4°C. The primary antibodies used were as follows: a pool of mouse anti-VN1203 HA monoclonal antibodies (15A3, 3G2, 7A11, 8A3, 14C5 and 18E1; Rockland); rabbit anti-K173 polyclonal antibody; rabbit anti-surfactant protein A polyclonal antibody (Millipore); and mouse anti-surfactant protein A monoclonal antibody (Abcam). Antibody binding was detected by using an IgG secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 633 (Molecular Probes). Sections were also counterstained with Hoechst 33342, trihydrochloride, trihydrate (Molecular Probes). The samples were examined by using confocal laser scanning microscopy (model LSM 510; Carl Zeiss).

To confirm sialic-acid-specific virus binding, tissue sections were treated, before incubation with viruses, with Arthrobacter ureafaciens sialidase (Sigma) for 3 h at 37°C. Viruses bound to tissue were detected as described above.

Experimental infection of ferrets

Animal studies were performed in accordance with the Animal Care and Use Committee guidelines of the University of Wisconsin-Madison. We used 6-10-month-old female ferrets (Triple F Farms) that were serologically negative by haemagglutination inhibition (HI) assay for currently circulating human influenza viruses. Six ferrets per group were anaesthetized intramuscularly with ketamine and xylazine (5-30 mg and 0.2-6 mg kg–1 of body weight, respectively) and inoculated intranasally with 106 p.f.u. (500 μl) of viruses. On days 3 and 6 after infection, three ferrets per group were killed for virological and pathological examinations. The virus titres in various organs were determined by use of plaque assays in MDCK cells.

Excised tissue samples of nasal turbinates, trachea, lungs, brain, liver, spleen, kidney and colon from euthanized ferrets were preserved in 10% phosphate-buffered formalin. Tissues were then trimmed and processed for paraffin embedding and cut into 5-μm-thick sections. One section from each tissue sample was stained by using a standard haematoxylin-and-eosin procedure, whereas another one was processed for immunohistological staining with a mixture of two anti-influenza virus rabbit antibodies (1:2,000; R309 and anti-VN1203; both prepared in our laboratory) that react with CA04 and VN1203, respectively. Specific antigen-antibody reactions were visualized by using an indirect two-step dextran-polymer technique (Dako EnVision system; Dako) and 3,3' diaminobenzidine tetrahydrochloride staining (Dako).

Ferret transmission study

For transmission studies in ferrets, animals were housed in adjacent transmission cages that prevented direct and indirect contact between animals but allowed spread of influenza virus through the air (Showa Science; Supplementary Fig. 7). Three, five, or six ferrets were inoculated intranasally with 106 p.f.u. (500 βl) of virus (inoculated ferrets). Twenty-four hours after infection, three, five, or six naive ferrets were each placed in a cage adjacent to an inoculated ferret (contact ferrets). The ferrets were monitored for changes in body weight and the presence of clinical signs. To assess viral replication in nasal turbinates, we determined viral titres in nasal washes collected from virus-inoculated and contact ferrets on day 1 after inoculation or co-housing, respectively, and then every other day.

Serological tests

Serum samples were collected between days 14 and 20 after infection, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by use of an HI assay with 0.5% TRBCs (http://www.wpro.who.int/entity/emerging_diseases/documents/docs/manualonanimalaidiagnosisandsurveillance.pdf). Viruses bearing homologous HA were used as antigens for the HI tests.

Polykaryon formation representing membrane fusion activity

Monolayers of HeLa cells grown in 12-well plates were transfected with the protein expression vector pCAGGS46 encoding wild-type or mutant HA. At 24 h after transfection, cells transiently expressing HA protein were treated with trypsin (1 μg ml–1) in MEM containing 0.3% BSA for 30 min at 37°C to cleave the HA into its HA1 and HA2 subunits. Polykaryon formation was inducedby exposing the cells to low-pH buffer (145 mM NaCl, 20 mM sodium citrate (pH 6.0-5.4)) for 2 min at 37°C. After this exposure, the low-pH buffer was replaced with MEM containing 10% FBS and the cells were incubated for 3 h at 37°C. The cells were then fixed with methanol and stained with Giemsa's solution and photographed with a digital camera mounted on an inverted microscope (Nikon, Eclipse Ti). For quantitative analyses, cell nuclei were counted in five randomly chosen fields of cell culture. Polykaryon formation activity was calculated from the number of nuclei in polykaryons divided by the total number of nuclei in the same field.

Thermostability

Viruses (128 HA units in PBS) were incubated for the times indicated at 50°C. Subsequently, infectivity and haemagglutination activity were determined by use of plaque assays in MDCK cells and haemagglutination assays using 0.5% TRBCs, respectively.

Neuraminidase (NA) inhibition assay

To assess the sensitivity of viruses to the NA inhibitor oseltamivir, NA inhibition assays were performed as described previously32.

Statistical analysis

All statistical analyses were performed using JMP 9.0.0 (SAS Institute Inc.). The statistical significance of differences between rgCA04 and H5 avian/human reassortant viruses was determined by using a Dunnett's test. Comparisons of polykaryon formation between wild-type and mutant HAs were done using Tukey's test. P values of <0.05 were considered significant.

46. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-199(1991).

REPORT

Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets

Sander Herfst,1 Eefje J. A. Schrauwen,1 Martin Linster,1 Salin Chutinimitkul,1 Emmie de Wit,1* Vincent J. Munster,1* Erin M. Sorrell,1 Theo M. Bestebroer,1 David F. Burke,2 Derek J. Smith,123 Guus F. Rimmelzwaan,1 Albert D. M. E. Osterhaus,1 Ron A. M. Fouchier1

Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet (“airborne transmission”) between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

Influenza A viruses have been isolated from many host species, including humans, pigs, horses, dogs, marine mammals, and a wide range of domestic birds, yet wildbirds in the orders Anseriformes (ducks, geese, and swans) and Charad-riiformes (gulls, terns, and waders) are thought to form the virus reservoir in nature (1). Influenza A viruses belong to the family Orthomyxoviridae; these viruses have an RNA genome consisting of eight gene segments (2, 3). Segments 1 to 3 encode the polymerase proteins: basic polymerase 2 (PB2), basic polymerase 1 (PB1), and acidic polymerase (PA), respectively. These proteins form the RNA-dependent RNA polymerase complex responsible for transcription and replication of the viral genome. Segment 2 also encodes a second small protein, PB1-F2, which has been implicated in the induction of cell death (4, 5). Segments 4 and 6 encode the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), respectively. HA is responsible for binding 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, responsible for cleaving SAs from host cells and virus particles. Segment 5 codes for the nucleo-capsid protein (NP) that binds to viral RNA and, together with the polymerase proteins, forms the ribonucleoprotein complexes (RNPs). Segment 7 codes for the viral matrix structural protein M1 and the ion-channel protein M2 that is incorporated in the viral membrane. Segment 8 encodes the nonstructural protein NS1 and the nucleic-export protein (NEP) previously known as NS2. NS1 is an antagonist of host innate immune responses and interferes with host gene expression, whereas NEP is involved in the nuclear export of RNPs into the cytoplasm before virus assembly (2, 3).

Influenza A viruses show pronounced genetic variation of the surface glycoproteins HA and NA (1). Consequently, the viruses are classified based on the antigenic variation of the HA and NA proteins. To date, 16 major antigenic variants of HA and 9 of NA have been recognized in wild birds and are found in numerous combinations designated as virus subtypes (for instance, H1N1, H5N1, H7N7, and H16N3), which are used in influenza A virus classification and nomenclature (1, 6). This classification system is biologically relevant, as natural host antibodies that recognize one HA or NA subtype will generally not cross-react with other HA and NA subtypes.

On the basis of their virulence in chickens, influenza A viruses of the H5 and H7 subtypes can be further classified into highly pathogenic avian influenza (HPAI) and low-pathogenic avian influenza (LPAI) viruses. Viruses of subtypes H1 to H4, H6, and H8 to H16 are LPAI viruses. The vast majority of H5 and H7 influenza A viruses are also of the LPAI phenotype. HPAI viruses are generally thought to arise in poultry after domestic birds become infected by LPAI H5 and H7 viruses from the wild-bird reservoir (7,8). The HA protein of influenza A viruses is initially synthesized as a single polypeptide precursor (HA0), which is cleaved into HA1 and HA2 sub-units by trypsin-like proteases in the host cell. The switch from LPAI to HPAI virus phenotype occurs upon the introduction of basic amino acid residues into the HA0 cleavage site, also known as the multibasic cleavage site (MBCS). The MBCS in HA can be cleaved by ubiquitously expressed host proteases; this cleavage facilitates systemic virus replication and results in mortality of up to 100% in poultry (9,10).

Since the late 1990s, HPAIA/H5N1 viruses have devastated the poultry industry of numerous countries in the Eastern Hemisphere. To date, A/H5N1 has spread from Asia to Europe, Africa, and the Middle East, resulting in the death of hundreds of millions of domestic birds. In Hong Kong in 1997, the first human deaths directly attributable to avian A/H5N1 virus were recorded (11). Since 2003, more than 600 laboratory-confirmed cases of HPAI A/H5N1 virus infections in humans have been reported from 15 countries (12). Although limited A/H5N1 virus transmission between persons in close contact has been reported, sustained human-to-human transmission of HPAI A/H5N1 virus has not been detected (13-15). Whether this virus may acquire the ability to be transmitted via aerosols or respiratory droplets among mammals, including humans, to trigger a future pandemic is a key question for pandemic preparedness. Although our knowledge of viral traits necessary for host switching and virulence has increased substantially in recent years (16,17), the factors that determine airborne transmission of influenza viruses among mammals, a trait necessary for a virus to become pandemic, have remained largely unknown (18-21). Therefore, investigations of routes of influenza virus transmission between animals and on the determinants of airborne transmission are high on the influenza research agenda.

The viruses that caused the major pandemics of the past century emerged upon reassortment (that is, genetic mixing) of animal and human influenza viruses (22). However, given that viruses from only four pandemics are available for analyses, we cannot exclude the possibility that a future pandemic may be triggered by a wholly avian virus without the requirement of reassortment. Several studies have shown that reassortment events between A/H5N1 and seasonal human influenza viruses do not yield viruses that are readily transmitted between ferrets (18-20, 23). In our work, we investigated whether A/H5N1 virus could change its transmissibility characteristics without any requirement for reassortment.

We chose influenza virus A/Indonesia/5/2005 for our study because the incidence of human A/H5N1 virus infections and fatalities in Indonesia remains fairly high (12), and there are concerns that this virus could acquire molecular characteristics that would allow it to become more readily transmissible between humans and initiate a pandemic. Because no reassortants between A/H5N1 viruses and seasonal or pandemic human influenza viruses have been detected in nature and because our goal was to understand the biological properties needed for an influenza virus to become airborne transmissible in mammals, we decided to use the complete A/Indonesia/5/2005 virus that was isolated from a human case of HPAI A/H5N1 infection.

We chose the ferret (Mustela putoiius faro) as the animal model for our studies. Ferrets have been used in influenza research since 1933 because they are susceptible to infection with human and avian influenza viruses (24). After infection with human influenza A virus, ferrets develop respiratory disease and lung pathology similar to that observed in humans. Ferrets can also transmit human influenza viruses to other ferrets that serve as sentinels with or without direct contact (fig. S1) (25-27).

Host restriction of replication and transmission of influenza A viruses is partly determined by specific SA receptors on the surface of susceptible cells. The affinity of influenza viruses for these receptors varies according to the species from which they are isolated. Influenza viruses of avian origin preferentially bind to α-2,3–linked SA receptors, whereas human influenza viruses recognize α-2,6-linked SA receptors. The receptor distribution in ferrets resembles that of humans in that the α-2,6-linked SA receptors are predominantly present in the upper respiratory tract (URT), and the α-2,3–linked SA receptors are mainly present in the lower respiratory tract. In chickens and other birds, α-2,3–linked SAs predominate, but both α-2,3–linked and α-2,6-linked SA are present throughout the respiratory and enteric tracts (fig. S2) (28). The differences in receptor distribution between humans and avian species are thought to determine the host restriction of influenza A viruses. A switch in receptor specificity from avian α-2,3-SA to human α-2,6-SA receptors, which can be acquired by specific mutations in the receptor binding site (RBS) of the HA, is expected to be necessary for an avian virus 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 already been shown to be major determinants of host range and transmission, including for pandemic influenza viruses (29-31). Whereas avian viruses, in principle, replicate at temperatures around 41°C (the temperature in the intestinal tract of birds), for replication in humans the viruses need to adapt to 33°C (the temperature of the human URT). The amino acid substitution Glu627→Lys627 (E627K) in the polymerase complex protein PB2 has been associated with increased virus replication in mammalian cells at such lower temperatures (16,17, 32).

In addition, when newly formed virus particles bud from the host cell membrane after virus replication, the NA present on the virus membrane facilitates the release of particles. For A/H5N1, this process is rather inefficient, and released particles tend to form virus aggregates (22). Therefore, a balance between the properties endowed by HA and NA may be required to generate single particles. These established effects were thus used as the basis for the initial substitutions chosen in the current study.

Human-to-human transmission of influenza viruses can occur through direct contact, indirect contact via fomites (contaminated environmental surfaces), and/or airborne transmission via small aerosols or large respiratory droplets. The pandemic and epidemic influenza viruses that have circulated in humans throughout the past century were all transmitted via the airborne route, in contrast to many other respiratory viruses that are exclusively transmitted via contact. There is no exact particle size cut-off at which transmission changes from exclusively large droplets to aerosols. However, it is generally accepted that for infectious particles with a diameter of 5 μm or less, transmission occurs via aerosols. Because we did not measure particle size during our experiments, we will use the term “airborne transmission” throughout this Report.

Biosafety and biosecurity concerns have remained foremost in our planning for this research program. The details are explained in the supplementary materials and are summarized here: The enhanced Animal Biosafety Laboratory level 3 (ABSL3+) facility at Erasmus Medical Center (MC) Rotterdam, the Netherlands, was constructed for the specific purpose of containing pathogenic and transmissible influenza viruses and other pathogens of concern. The facility consists of a negatively pressurized laboratory with an interlock room. All in vivo and in vitro experimental work is carried out in negatively pressurized class 3 isolators or class 3 biosafety cabinets, respectively. The facility is secured by procedures recognized as appropriate by the institutional biosafety officers and facility management at Erasmus MC, as well as Dutch and U.S. government inspectors.

Before and during the research, biosafety officers of Erasmus MC and inspectors from the Dutch government, as well as from the U.S. Centers for Disease Control and Prevention, approved the facilities and procedures. Explicit permits for research on genetically modified airborne-transmissible A/H5N1 virus were obtained from the Dutch government. The research was performed strictly in accordance with the Dutch Code of Conduct for Biosecurity (33). All personnel were instructed and trained extensively for working in the ABSL3+ facility, handling (highly pathogenic) influenza virus, and controlling incidents (such as spills). To further prevent occupational risks, research personnel used protective equipment and were offered seasonal and A/H5N1 influenza vaccines (25). For emergency purposes, Erasmus MC holds supplies of oseltamivir and has quarantine hospital rooms.

Using a combination of targeted mutagenesis followed by serial virus passage in ferrets, we investigated whether A/H5N1 virus can acquire mutations that would increase the risk of mammalian transmission (34). We have previously shown that several amino acid substitutions in the RBS of the HA surface glycoprotein of A/Indonesia/5/2005 change the binding preference from the avian α-2,3–linked SA receptors to the human α-2,6-linked SA receptors (35). A/Indonesia/5/2005 virus with amino acid substitutions N182K, Q222L/G224S, or N182K/Q222L/G224S (numbers refer to amino acid positions in the mature H5 HA protein; N, Asn; Q, Gln; L, Leu; G, Gly; S, Ser) in HA display attachment patterns similar to those of human viruses to cells of the respiratory tract of ferrets and humans (35). Of these changes, we know that together, Q222L and G224S switch the receptor binding specificity of H2 and H3 subtype influenza viruses, as this switch contributed to the emergence of the 1957 and 1968 pandemics (36). N182K has been found in a human case of A/H5N1 virus infection (37).

Our experimental rationale to obtain transmissible A/H5N1 viruses was to select a mutant A/H5N1 virus with receptor specificity for α-2,6-linked SA shed at high titers from the URT of ferrets. Therefore, we used the QuickChange multisite-directed mutagenesis kit (Agilent Technologies, Amstelveen, the Netherlands) to introduce amino acid substitutions N182K, Q222L/G224S, or N182K/Q222L/G224S in the HA of wild-type (WT) A/Indonesia/5/2005, resulting in A/H5N1HA N182K, A/H5N1HA Q222L, G224S, and A/H5N1HA N182K, Q222L, G224S. Experimental details for experiments 1 to 9 are provided in the supplementary materials (25). For experiment 1, we inoculated these mutant viruses and the A/H5N1wildtype virus intranasally into groups of six ferrets for each virus (fig. S3). Throat and nasal swabs were collected daily, and virus titers were determined by end-point dilution in Madin Darby canine kidney (MDCK) cells to quantify virus shedding from the ferret URT. Three animals were euthanized after day 3 to enable tissue sample collection. All remaining animals were euthanized by day 7 when the same tissue samples were taken. Virus titers were determined in the nasal turbinates, trachea, and lungs collected postmortem from the euthanized ferrets. Throughout the duration of experiment 1, ferrets inoculated intranasally with A/H5N1wildtype virus produced high titers in nose and throat swabs—up to 10 times more than A/H5N1HA Q222L, G224S, which yielded the highest virus titers of all three mutants during the 7-day period (Fig. 1). However, no significant difference was observed between the virus shedding of ferrets inoculated with A/H5N1HA Q222L, G224S or A/H5N1HA N182K during the first 3 days when six animals per group were present. Thus, of the viruses with specificity for α-2,6-linked SA, A/H5N1HA Q222L, G224S yielded the highest virus titers in the ferret URT (Fig. 1).

Fig. 1. In experiment 1, we inoculated groups of six ferrets intranasally with 1 × 106 TCID50 of (A) influenza A/H5N1wildtype virus and the three mutants (B) A/H5N1HA N182K, (C) A/H5N1HA Q222L, G224S, and (D) A/H5N1HA N182K, Q222L, G224S.

Fig. 1

In experiment 1, we inoculated groups of six ferrets intranasally with 1 × 106 TCID50 of (A) influenza A/H5N1wildtype virus and the three mutants (B) A/H5N1HA N182K, (C) A/H5N1HA Q222L, G224S, and (D) A/H5N1HA N182K, Q222L, G224S. Three animals (more...)

As described above, amino acid substitution E627K in PB2 is one of the most consistent host-range determinants of influenza viruses (29-31). For experiment 2 (fig. S4), we introduced E627K into the PB2 gene of A/Indonesia/5/2005 by site-directed mutagenesis and produced the recombinant virus A/H5N1HA Q222L, G224S PB2 E627K The introduction of E627K in PB2 did not significantly affect virus shedding in ferrets, because virus titers in the URT were similar to those seen in A/H5N1HA Q222L, G224S-inoculated animals [up to 1 × 104 50% tissue culture infectious doses (TCID50)] (Mann-Whitney U rank-sum test, P = 0.476) (Fig. 1 and fig. S5). When four naïve ferrets were housed in cages adjacent to those with four inoculated animals to test for airborne transmission as described previously (27), A/H5N1HA Q222L, G224S PB2 E627K was not transmitted (fig. S5).

Because the mutant virus harboring the E627K mutation in PB2 and Q222L and G224S in HA did not transmit in experiment 2, we designed an experiment to force the virus to adapt to replication in the mammalian respiratory tract and to select virus variants by repeated passage (10 passages in total) of the constructed A/H5N1HA Q222L, G224S PB2 E627K virus and A/H5N1wiidtype virus in the ferret URT (Fig. 2 and fig. S6). In experiment 3, one ferret was inoculated intranasally with A/H5N1wildtype and one ferret with A/H5N1HA Q222L, G224S PB2 E627K. Throat and nose swabs were collected daily from live animals until 4 days postinoculation (dpi), at which time the animals were euthanized to collect samples from nasal turbinates and lungs. The nasal turbinates were homogenized in 3 ml of virus-transport medium, tissue debris was pelleted by centrifugation, and 0.5 ml of the supernatant was subsequently used to inoculate the next ferret intranasally (passage 2). This procedure was repeated until passage 6.

Fig. 2. Experiment 3, virus passaging in ferrets (P1 to P10, passages 1 to 10).

Fig. 2

Experiment 3, virus passaging in ferrets (P1 to P10, passages 1 to 10). Because no airborne transmission was observed in experiment 2, A/H5N1wildtype and A/H5N1HA Q222L, G224S PB2 E627K were serially passaged in ferrets to allow adaptation for efficient (more...)

From passage 6 onward, in addition to the samples described above, a nasal wash was also collected at 3 dpi. To this end, 1 ml of phosphate-buffered saline (PBS) was delivered dropwise to the nostrils of the ferrets to induce sneezing. Approximately 200 μl of the “sneeze” was collected in a Petri dish, and PBS was added to a final volume of 2 ml. The nasal-wash samples were used for intranasal inoculation of the ferrets for the subsequent passages 7 through 10. We changed the source of inoculum during the course of the experiment, because passaging nasal washes may facilitate the selection of viruses that were secreted from the URT. Because influenza viruses mutate rapidly, we anticipated that 10 passages would be sufficient for the virus to adapt to efficient replication in mammals.

Virus titers in the nasal turbinates of ferrets inoculated with A/H5N1wildtype ranged from~1 × 105 to 1× 107 TCID50/gram tissue throughout 10 serial passages (Fig. 3A and fig. S7). In ferrets inoculated with A/H5N1HA Q222L, G224S PB2 E627K virus, a moderate increase in virus titers in the nasal turbinates was observed as the passage number increased. These titers ranged from 1 × 104 TCID50/gram tissue at the start of the experiment to 3.2× 105 to 1 × 106 TCID50/gram tissue in the final passages (Fig. 3A and fig. S7). Notably, virus titers in the nose swabs of animals inoculated with A/H5N1HA Q222L, G224S PB2 E627K also increased during the successive passages, with peak virus shedding of 1 × 105 TCID50 at 2 dpi after 10 passages (Fig. 3B). These data indicate that A/H5N1HA Q222L, G224S PB2 E627K was developing greater capacity to replicate in the ferret URT after repeated passage, with evidence for such adaptation becoming apparent by passage number 4. In contrast, virus titers in the nose swabs of the ferrets collected at 1 to 4 dpi throughout 10 serial passages with A/H5N1wildtype revealed no changes in patterns of virus shedding.

Fig. 3. Virus titers in (A) the nasal turbinates collected at day 4 and (B) nose swabs collected daily until day 4, from ferrets inoculated with A/H5N1wildtype (blue) and A/H5N1HA Q222L, G224S PB2 E627K (red) throughout the 10 serial passages described in Fig.

Fig. 3

Virus titers in (A) the nasal turbinates collected at day 4 and (B) nose swabs collected daily until day 4, from ferrets inoculated with A/H5N1wildtype (blue) and A/H5N1HA Q222L, G224S PB2 E627K (red) throughout the 10 serial passages described in Fig. (more...)

Passaging of influenza viruses in ferrets should result in the natural selection of heterogeneous mixtures of viruses in each animal with a variety of mutations: so-called viral quasi-species (38). The genetic composition of the viral quasi-species present in the nasal washes of ferrets after 10 passages of A/H5N1wildtype and A/H5N1HA Q222L, G224S PB2 E627K was determined by sequence analysis using the 454/Roche GS-FLX sequencing platform (Roche, Woerden, the Netherlands) (tables S1 and S2). The mutations introduced in A/H5N1HA Q222L, G224S PB2 E627K by reverse genetics remained present in the virus population after 10 consecutive passages at a frequency >99.5% (Fig. 4 and table S1). Numerous additional nucleotide substitutions were detected in all viral gene segments of A/H5N1wildtype and A/H5N1HA Q222L, G22L PB2 E627K after passaging, except in segment 7 (tables S1 and S2). Of The 30 nucleotide substitutions selected during serial passage, 53% resulted in amino acid substitutions. The only amino acid substitution detected upon repeated passage of both AH5N1wildtype and AH5N1HA Q222L, G224S PB2 E627K was T156A (T, Thr; A, Ala) in HA. This substitution removes a potential N-linked glycosylation site (Asn-X-Thr/Ser; X, any amino acid) in HA and was detected in 99.6% of the A/HENU1wildtype sequences after 10 passages. T156A was detected in 89% of the A/H5N1HA Q222L, G224S PB2 E627K sequences after 10 passages, and the other 11% of sequences possessed the substitution N154K, which removes the same potential N-linked glycosylation site in HA.

Fig. 4. Summary of the substitutions detected upon serial passage and airborne transmission of A/H5N1HA Q222L, G224S PB2 E627K virus in ferrets.

Fig. 4

Summary of the substitutions detected upon serial passage and airborne 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, (more...)

In experiment 4 (see supplementary materials), we investigated whether airborne-transmissible viruses were present in the heterogeneous virus population generated during virus passaging in ferrets (fig. S4). Nasal-wash samples, collected at 3 dpi from ferrets at passage 10, were used in transmission experiments to test whether airborne-transmissible virus was present in the virus quasi-species. For this purpose, nasal-wash samples were diluted 1:2 in PBS and subsequently used to inoculate six naïve ferrets intranasally: two for passage 10 A/H5N1wildtype and four for passage 10 A/H5N1HA Q222L, G224S PB2 E627K virus.

The following day, a naïve recipient ferret was placed in a cage adjacent to each inoculated donor ferret. These cages are designed to prevent direct contact between animals but allow airflow from a donor ferret to a neighboring recipient ferret (fig. S1) (27). Although mutations had accumulated in the viral genome after passaging of A/H5N1wildtype in ferrets, we did not detect replicating virus upon inoculation of MDCK cells with swabs collected from naïve recipient ferrets after they were paired with donor ferrets inoculated with passage 10 A/H5N1wildtype virus (Fig. 5, A and B). In contrast, we did detect virus in recipient ferrets paired with those inoculated with passage 10 A/H5N1HA Q222L, G224S PB2 E627K virus. Three (F1 to F3) out of four (F1 to F4) naïve recipient ferrets became infected as confirmed by the presence of replicating virus in the collected nasal and throat swabs (Fig. 5, C and D). A throat-swab sample obtained from recipient ferret F2, which contained the highest virus titer among the ferrets in the first transmission experiment, was subsequently used for intranasal inoculation of two additional donor ferrets. Both of these animals, when placed in the transmission cage setup (fig. S1), again transmitted the virus to the recipient ferrets (F5 and F6) (Fig. 6, A and B). A virus isolate was obtained after inoculation of MDCK cells with a nose swab collected from ferret F5 at 7 dpi. The virus from F5 was inoculated intranasally into two more donor ferrets. One day later, these animals were paired with two recipient ferrets (F7 and F8) in transmission cages, one of which (F7) subsequently became infected (Fig. 6, C and D).

Fig. 5. Airborne transmission of A/H5N1 viruses in ferrets.

Fig. 5

Airborne transmission of A/H5N1 viruses in ferrets. Transmission experiments are shown for A/H5N1Wildtype (A and B) and A/H5N1HA Q222L, G224S PB2 E627K (C and D) after 10 passages (P10) in ferrets. Two or four ferrets were inoculated intranasally with nasal-wash (more...)

Fig. 6. Comparison of airborne transmission of experimental passaged A/H5N1 and 2009 pandemic A/H1N1 viruses in individual ferrets.

Fig. 6

Comparison of airborne transmission of experimental passaged A/H5N1 and 2009 pandemic A/H1N1 viruses in individual ferrets. A throat-swab sample from ferret F2 at 7 days postexposure (dpe) (Fig. 5D) was used for the transmission experiments shown in (more...)

We used conventional Sanger sequencing to determine the consensus genome sequences of viruses recovered from the six ferrets (F1 to F3 and F5 to F7) that acquired virus via airborne transmission (Fig. 4 and table S3). All six samples still harbored substitutions Q222L, G224S, and E627K that had been introduced by reverse genetics. Surprisingly, only two additional amino acid substitutions, both in HA, were consistently detected in all six airborne-transmissible viruses: (i) H103Y (H, His; Y, Tyr), which forms part of the HA trimer interface, and (ii) T156A, which is proximal but not immediately adjacent to the RBS (fig. S8). Although we observed several other mutations, their occurrence was not consistent among the airborne viruses, indicating that of the heterogeneous virus populations generated by passaging in ferrets, viruses with different genotypes were transmissible. In addition, a single transmission experiment is not sufficient to select for clonal airborne-transmissible viruses because, for example, the consensus sequence of virus isolated from F6 differed from the sequence of parental virus isolated from F2.

Together, these results suggest that as few as five amino acid substitutions (four in HA and one in PB2) may be sufficient to confer airborne transmission of HPAI A/H5N1 virus between mammals. The airborne-transmissible virus isolate with the least number of amino acid substitutions, compared with the A/H5N1wildtype, was recovered from ferret F5. This virus isolate had a total of nine amino acid substitutions; in addition to the three mutations that we introduced (Q222L and G224S in HA and E627K in PB2), this virus harbored H103Y and T156A in HA, H99Y and I368V (I, Ile; V, Val) in PB1, and R99K (R, Arg) and S345N in NP (table S3). Reverse genetics will be needed to identify which of the five to nine amino acid substitutions in this virus are essential to confer airborne transmission.

During the course of the transmission experiments with the airborne-transmissible viruses, ferrets displayed lethargy, loss of appetite, and ruffled fur after intranasal inoculation. One of eight inoculated animals died upon intranasal inoculation (Table 1). In previously published experiments, ferrets inoculated intranasally with WTA/Indonesia/5/2005 virus at a dose of 1 × 106 TCID50 showed neurological disease and/or death (39,40). It should be noted that inoculation of immunologically naïve ferrets with a dose of 1 × 106 TCID50 of A/H5N1 virus and the subsequent course of disease is not representative of the natural situation in humans. Importantly, although the six ferrets that became infected via respiratory droplets or aerosol also displayed lethargy, loss of appetite, and ruffled fur, none of these animals died within the course of the experiment. Moreover, previous infections of humans with seasonal influenza viruses are likely to induce heterosubtypic immunity that would offer some protection against the development of severe disease (41,42). It has been shown that mice and ferrets previously infected with an A/H3N2 virus are clinically protected against intranasal challenge infection with an A/H5N1 virus (43, 44).

Table 1. Lethality of WT and airborne-transmissible A/H5N1 virus in ferrets upon inoculation via different routes. n, number of animals; N.A., not applicable.

Table 1

Lethality of WT and airborne-transmissible A/H5N1 virus in ferrets upon inoculation via different routes. n, number of animals; N.A., not applicable.

After intratracheal inoculation (experiment 5; fig. S9), six ferrets inoculated with 1 × 106 TCID50 of airborne-transmissible virus F5 in a 3-ml volume of PBS died or were moribund at day 3. Intratracheal inoculations at such high doses do not represent the natural route of infection and are generally used only to test the ability of viruses to cause pneumonia (45), as is done for vaccination-challenge studies. At necropsy, the six ferrets revealed macroscopic lesions affecting 80 to 100% of the lung parenchyma with average virus titers of 7.9 × 106 TCID50/gram lung (fig. S10). These data are similar to those described previously for A/H5N1wildtype in ferrets (Table 1). Thus, although the airborne-transmissible virus is lethal to ferrets upon intratracheal inoculation at high doses, the virus was not lethal after airborne transmission.

To test the effect of the mutations in HA in the airborne-transmissible virus on its sensitivity to antiviral drugs, we used virus isolated from F5 (experiment 6). This airborne-transmissible virus with nine amino acid substitutions displayed a sensitivity to the antiviral drug oseltamivir similar to that of A/H5N1wildtype (table S4).

In experiment 7, we evaluated the recognition of the airborne-transmissible virus by antisera raised against potential A/H5N1 vaccine strains. Because only HA recognition by antibodies is evaluated in this assay, chimeric viruses were generated based on six gene segments of the mouse-adapted A/Puerto Rico/8/34 (PR8) virus with the HA and PB2 genes of the transmissible virus harboring amino acid substitutions H103Y, T156A, Q222L, and G224S in HA and E627K in PB2. We replaced the MBCS of the HA by a monobasic cleavage site, allowing us to do these experiments under BSL2 conditions. The chimeric PR8/H5 virus reacted well with ferret antisera raised against A/Indonesia/5/2005 and several other prepandemic vaccine strains (table S5). In fact, the presence of the four HA mutations increased the reactivity with H5 antisera by twofold or more.

We subsequently used the same PR8/H5 chimeric virus in experiment 8 to evaluate the presence of existing immunity against the airborne-transmissible virus in sera obtained from human volunteers more than 70 years of age. The introduction of receptor-binding site mutations Q222L/G224S and the mutations H103Y and T156A in HA, acquired during ferret passage, did not result in increased cross-reactivity with human antisera (table S6), indicating that humans do not have antibodies against the HA of the airborne-transmissible A/H5N1 virus that was selected in our experiments.

Substitutions Q222L and G224S have previously been shown to be sufficient to switch receptor-binding specificity of avian influenza strains (i.e., α-2,3–linked SA) to that of human strains (i.e., α-2,6-linked SA) (20, 35, 46, 47). Amino acid position 103 is distal from the RBS, forms part of the trimer interface, and is unlikely to affect receptor specificity (fig. S8). T156 is part of a N-glycosylation sequon, and T156A (as well as N154K) would delete this potential glycosylation site (fig. S8); amino acid T156 is proximal but not immediately adjacent to the RBS. Loss of N-glycosylation sites at the tip of HA has been shown to affect receptor binding of A/H1 (48,49) and the virulence of A/H5 virus (50). We evaluated the impact of the HA mutations that emerged during passaging in ferrets in a modified turkey red blood cell (TRBC) assay (Table 2). In this assay, the binding of influenza viruses, with a mutated HA, to normal TRBCs (expressing both α-2,3–linked SA and α-2,6-linked SA) and modified TRBCs with either α-2,3–linked SA or α-2,6-linked SA on the cell surface was evaluated and compared to two reference viruses with known receptor binding preference: avian A/H5N1 and human A/H3N2 viruses. As expected and shown before, introduction of the Q222L and G224S mutations in the HA of A/H5N1 changed the receptor binding preference from α-2,3–linked SA to α-2,6-linked SA (35). Furthermore, in our hands, the introduction of substitutions H103Yand T156A not only enhanced binding of A/H5N1HA Q222L, G224S PB2 E627K to α-2,6-linked SA, as expected from glycan array studies (51), but also increased the affinity for α-2,3–linked SA. When these two mutations were introduced in the A/H5N1wildtype HA, the affinity for α-2,3–linked SA also increased.

Substitutions Q222L and G224S have previously emerged in avian A/H2 and A/H3 viruses in nature (36, 52), and mutations associated with similar changes in receptor binding specificity have been detected repeatedly in A/H5 viruses—for instance, substitution N182K has been reported nine times (37, 51), which is why we initially selected it for our investigations. The other three substitutions we found consistently in airborne-transmissible viruses have all previously been detected in HPAI A/H5N1 viruses circulating in the field (53). Only a minor fraction of the A/H5N1 viruses that have circulated in outbreaks have been sequenced (estimated to be <0.001%) (53, 54). Yet the individual substitutions we obtained, as well as combinations of T156A and H103Yor T156A and E627K, have already been reported in public sequence databases (53); thus, we conclude that these mutations do not appear to have a detrimental effect on virus fitness. Substitution H103Y has only been found once, in combination with T156A in a duck in China (53). Substitution E627K in PB2 has been found in ~27% of avian A/H5N1 virus sequences and in ~29% of human A/H5N1 viruses (53). Substitution T156A in HA has been reported in >50% of the viruses sequenced and was detected in 100% of the viruses from human cases in Egypt (53).

Investigations of viral quasi-species during a massive avian influenza A/H7N7 virus outbreak in the Netherlands indicated that viruses with human adaptation markers, including HA mutations that alter receptor specificity and mutations in polymerase proteins that increase polymerase activity like E627K in PB2, emerged rapidly in poultry (55-57). Given the large numbers of HPAI A/H5N1 virus-infected hosts globally, the high viral mutation rate, and the apparent lack of detrimental effects on fitness of the mutations that confer airborne transmission, it may simply be a matter of chance and time before a human-to-human transmissible A/H5N1 virus emerges.

The specific mutations we identified in these experiments that are associated with airborne transmission represent biological traits that may be determined by a set of different amino acid substitutions. For example, amino acid substitutions D701N (D, Asp) or S590G/R591Q in PB2 yield a similar phenotype to E627K (29). N182K and other substitutions in the RBS of HA may yield a similar phenotype to Q222L/G224S (35). Such mutations should be considered for A/H5N1 surveillance studies in outbreak areas. Imai et al. recently identified different RBS changes (N220K, Q222L) along with N154D (affecting the same N-glycosylation sequon as T156A) and T314I in HA as determinants of airborne transmission of an A/H5 virus (58). This airborne virus contained seven genes of the 2009 pandemic A/H1N1 virus (which has S590G/R591Q in PB2 rather than E627K), with the HA of A/H5N1 virus A/Vietnam/1203/2004 (58). These data indicate that different lineages of A/H5N1 virus and different amino acid substitutions that affect particular biological traits (receptor binding, glycosylation, replication) can yield airborne-transmissible A/H5N1 viruses.

Although our experiments showed that A/H5N1 virus can acquire a capacity for airborne transmission, the efficiency of this mode remains unclear. Previous data have indicated that the 2009 pandemic A/H1N1 virus transmits efficiently among ferrets and that naïve animals shed high amounts of virus as early as 1 or 2 days after exposure (27). When we compare the A/H5N1 transmission data with that of reference (27), keeping in mind that our experimental design for studying transmission is not quantitative, the data shown in Figs. 5 and 6 suggest that A/H5N1 airborne transmission was less robust, with less and delayed virus shedding compared with pandemic A/H1N1 virus.

Airborne transmission could be tested in a second mammalian model system such as guinea pigs (59), but this would still not provide conclusive evidence that transmission among humans would occur. The mutations we identified need to be tested for their effect on transmission in other A/H5N1 virus lineages (60), and experiments are needed to quantify how they affect viral fitness and virulence in birds and mammals. For pandemic preparedness, antiviral drugs and vaccine candidates against airborne-transmissible virus should be evaluated in depth. Mechanistic studies on the phenotypic traits associated with each of the identified amino acid substitutions should provide insights into the key determinants of airborne virus transmission. Our findings indicate that HPAI A/H5N1 viruses have the potential to evolve directly to transmit by aerosol or respiratory droplets between mammals, without reassortment in any intermediate host, and thus pose a risk of becoming pandemic in humans. Identification of the minimal requirements for virus transmission between mammals may have prognostic and diagnostic value for improving pandemic preparedness (34).

References and Notes

Acknowledgments

We thank D. de Meulder, G. van Amerongen, and D. Akkermans for technical assistance. M. Peiris, Univ. of Hong Kong, provided A/Indonesia/5/2005 with permission from I. Kandun of the Indonesian government. This work was financed through NIAID-NIH contract HHSN266200700010C. D.J.S. and D.F.B. were supported in part by NIH Director's Pioneer Award DP1-OD000490-01. We acknowledge a Nederlandse Organisatie voor Wetenschappelijk Onderzoek VICI grant, European Union FP7 program EMPERIE (223498), and Human Frontier Science Program grant P0050/2008. D.F.B. and D.J.S. acknowledge the use of the CamGrid distributed computing resource. Sequence data generated from this study were deposited in GenBank with accession numbers CY116643 to CY116698. Special arrangements are in place with the NIH and the contractor at Mount Sinai School of Medicine, New York, for sharing the viruses (and plasmids) in the present paper; please contact R.A.M.F. A.D.M. E.O. and G.F.R. are CSO and part-time employee of ViroClinics Biosciences BV. A.D.M.E.O. has advisory affiliations on behalf of Viroclinics Biosciences BV with GlaxoSmithKline, Novartis, and Roche. A.D.M.E.O. and R.A.M.F. are holders of certificates of shares in ViroClinics Biosciences B.V. To avoid any possible conflict of interests, Erasmus MC policy dictates that the shares as such are held by the Stichting Administratiekantoor Erasmus Personeelsparticipaties. The board of this foundation is appointed by the Board of Governors of the Erasmus MC and exercises all voting rights with regard to these shares.

Supplementary Materials

www.sciencemag.org/cgi/content/full/336/6088/1534/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S10

Tables S1 to S6

References (61-72)

30 August 2011; accepted 31 May 2012

10.1126/science.1213362

Footnotes

1

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

2

ERATO infection-induced Host Responses Project, Saitama 332-0012, Japan.

3

Department of Special Pathogens, international Research Centerfor infectious Diseases, institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

4

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

5

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

6

Health Science Hills, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan.

1

Department of Virology, Erasmus Medical Center, Rotterdam, The Netherlands.

2

Department of Zoology, University of Cambridge, Cambridge, UK.

3

Fogarty International Center, National Institutes of Health (NIH), Bethesda, MD 20892, USA.

To whom correspondence should be addressed. E-mail: r​.fouchie@erasmusmc.nl

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25

See materials and methods and other supplementary materials on Science Online.

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Copyright 2013 by the National Academy of Sciences. All rights reserved.
Bookshelf ID: NBK206985

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