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LETTERdoi:10.1038/nature10831

Experimental adaptation of an influenza H5 HAconfers respiratory droplet transmission to areassortant H5 HA/H1N1 virus in ferretsMasaki 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 occasionallyinfect humans, but currently do not transmit efficiently amonghumans. The viral haemagglutinin (HA) protein is a knownhost-range determinant as it mediates virus binding to host-specific cellular receptors1–3. Here we assess the molecular changesin HA that would allow a virus possessing subtype H5 HA to betransmissible among mammals. We identified a reassortant H5HA/H1N1 virus—comprising H5 HA (from an H5N1 virus) withfour mutations and the remaining seven gene segments from a2009 pandemic H1N1 virus—that was capable of droplet transmis-sion in a ferret model. The transmissible H5 reassortant viruspreferentially recognized human-type receptors, replicated effi-ciently in ferrets, caused lung lesions and weight loss, but wasnot highly pathogenic and did not cause mortality. These resultsindicate that H5 HA can convert to an HA that supports efficientviral transmission in mammals; however, we do not know whetherthe four mutations in the H5 HA identified here would render awholly avian H5N1 virus transmissible. The genetic origin of theremaining seven viral gene segments may also critically contributeto transmissibility in mammals. Nevertheless, as H5N1 virusescontinue to evolve and infect humans, receptor-binding variantsof H5N1 viruses with pandemic potential, including avian–humanreassortant viruses as tested here, may emerge. Our findingsemphasize the need to prepare for potential pandemics caused byinfluenza viruses possessing H5 HA, and will help individuals con-ducting surveillance in regions with circulating H5N1 viruses torecognize key residues that predict the pandemic potential of iso-lates, which will inform the development, production and distri-bution of effective countermeasures.

Although H5N1 viruses continue to cause outbreaks in poultry andthere are cases of human infection in Indonesia, Vietnam, Egypt andelsewhere (http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html), they have not acquired theability to cause human-to-human transmission. Investment in H5N1vaccines has therefore been questioned. However, because humanslack immunity to influenza viruses possessing an H5 HA, the emergenceof a transmissible H5-HA-possessing virus would probably cause apandemic. To prepare better for such a scenario, it is critical that weunderstand the molecular changes that may render H5-HA-possessingviruses transmissible in mammals. Such knowledge would allow us tomonitor circulating or newly emerging variants for their pandemicpotential, focus eradication efforts on viruses that already haveacquired subsets of molecular changes critical for transmission inmammals, stockpile antiviral compounds in regions where such virusescirculate, and initiate vaccine generation and large-scale production

before a pandemic. Therefore, we studied the molecular features thatwould render H5-HA-possessing viruses transmissible in mammals.

Previous studies suggested that HA has a major role in host-rangerestriction of influenza A viruses1–3. The HA of human isolates preferen-tially recognizes sialic acid linked to galactose by a2,6-linkages(Siaa2,6Gal), whereas the HA of avian isolates preferentially recognizessialic acid linked to galactose by a2,3-linkages (Siaa2,3Gal)3. A smallnumber of avian H5N1 viruses isolated from humans show limitedbinding to human-type receptors, a property conferred by several aminoacid changes in HA4–9. None of the H5N1 viruses tested transmittedefficiently in a ferret model10–13, although, while our paper was underreview, one study14 reported that a virus with a mutant H5 HA and aneuraminidase (NA) of a human virus in the H5N1 virus backgroundcaused 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 mutationsinto the globular head (amino acids 120–259 (H3 numbering), whichincludes the receptor-binding pocket) of A/Vietnam/1203/2004(H5N1; VN1203) HA (Supplementary Fig. 1). Although this viruswas isolated from a human, its HA retains avian-type receptor-bindingproperties6,15. We also replaced the multibasic HA cleavage sequencewith a non-virulent-type cleavage sequence, allowing us to per-form studies in biosafety level 2 containment (http://www.who.int/csr/resources/publications/influenza/influenzaRMD2003_5.pdf). Themutated polymerase chain reaction (PCR) products were cloned intoRNA polymerase I plasmids16 containing the VN1203 HA comple-mentary DNA, which resulted in Escherichia coli libraries representingthe randomly generated HA variants. Sequence analysis of 48randomly selected clones indicated an average of 1.0 amino acidchanges per HA globular head (data not shown). To generate anH5N1 virus library, plasmids for the synthesis of the mutated HA geneand the unmodified NA gene of VN1203 were transfected into humanembryonic kidney (293T) cells together with plasmids for the synthesisof the six remaining viral genes of A/Puerto Rico/8/34 (H1N1; PR8), alaboratory-adapted human influenza A virus.

Turkey red blood cells (TRBCs; which possess both Siaa2,6Gal andSiaa2,3Gal on their surface (data not shown)) were treated withSalmonella enterica serovar Typhimurium LT2 sialidase, which pref-erentially removes a2,3-linked sialic acid (that is, avian-type receptors),creating TRBCs that predominantly possess Siaa2,6Gal on the cellsurface (Siaa2,6-TRBCs; Supplementary Fig. 2). The virus librarywas then adsorbed to Siaa2,6-TRBCs at 4 uC and extensively washedto remove nonspecifically or weakly bound viruses. Bound viruses wereeluted by incubation at 37 uC for 30 min, and then diluted to approxi-mately ,0.5 viruses per well (on the basis of a pilot experiment that

1Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. 2ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan. 3Departmentof Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 4Department of Microbiology and Infectious Diseases,Kobe University, Hyogo 650-0017, Japan. 5Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 6Health ScienceHills, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan.

0 0 M O N T H 2 0 1 2 | V O L 0 0 0 | N A T U R E | 1

Macmillan Publishers Limited. All rights reserved©2012

ƽěG£ƾȒɉȵɆȔɉȜɎH5N1ɏǼȻ�ƷǺ>čĝ§Ýŕ:Ȏ³DzǟȊǻǢɝReceived 18 August 2011; Accepted 09 March 2012; Published online 02 May 2012; Updated online 22 May 2012

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