P-9

Receptor binding specificity of highly pathogenic
avian influenza viruses:
Development of improved assay system to determine the mutated
H5 avian viruses that acquire human receptor specificity
Yasuo Suzuki1,2 and Hiroaki Hiramatsu1
College of Life and Health Sciences and Health Science Hills, Chubu University;
2
Global COE Program, School of Pharmaceutical Sciences, University of Shizuoka
1Influenza is one of the most broadly spread zoonotic infectious diseases in theworld, and its pathogen, the influenza virus, is extremely mutable. Currently, highly
pathogenic avian influenza (subtype H5N1), which is continuing to spread across Asia,Europe, and the African continent, has already been transmitted to humans and has been
undergoing possible mutation for propagation among humans. When this new variant of
the virus becomes an epidemic among humans, it will spread throughout the world
within a short time resulting in a pandemic because humans do not have immunity tothe virus.
Most avian influenza viruses bind preferentially to sialic acid (Sia)α2-3Galctose(Gal) (avian-type receptor), whereas human influenza viruses bind preferentially toSiaα2-6Gal (human-type receptor). Switch from Siaα2-3Gal to Siaα2-6Gal receptor
specificity is a critical step in the adaptation of avian viruses to human hosts.
We (1-4) have detected that mutations of single or set of few amino acids atpositions in the hemagglutinins (HAs) of H5N1 which are isolated in China, Vietnamand Thailand independently converted the virus known to recognize the avian receptor
to ones that recognize the human receptor. Using the reverse genetics technique, we alsoidentified the potential amino acids in HA of H5N1 which involved in the mutations
responsible for the binding to human-type receptor. However, the assay system used inthese reports takes relatively long time (for 2-3days) and needs high amount of viruses.
In this report, we developed improved assay system to determine the mutated avian
influenza viruses that acquire human receptor specificity.
This method included coating of synthetic N-acetylneuraminic acidα2-3 (or 6)
Gal-glycopolymer synthesized from γ-polyglutamic acid, a component of traditional
Japanese food, “Natto” (5) to the 96 well plate, virus binding to the polymer, fix thebound virus with formalin, trapping the virus with 1st antibody directed the virus,trapping the 1st antibody with 2nd antibody linked with the enzyme, and detection of thecolor developed with enzyme substrate. It takes for 6 hrs to determine the receptor
binding specificity of the viruses that bind to avian- and human-type receptor with small
amount of virus (8HAU) in culture fluid or allantoic fluid of virus-infected cells andembryonated chicken eggs, respectively. This method needs no expensive machine andmay be useful to find the molecular signal of H5N1 avian viruses that acquire
human-type receptor specificity and pandemic potential.
1) Yasuo Suzuki, Experimental Glycoscience (Glycobiology) Springer pp. 225-2
26 (2008).
2) Nongluk Sriwilaijaroen et al., Glycoconjugate J., in press (2008)
3) Prasert Auewarakul et al., J. Virol., 81, 9950-9955 (2007)
4) Sinya Yamada, et al., Nature, 444, 378-382, (2006)5) M. Ogata et al., Bioorg. Med. Chem., 15 (3), 1383-1393 (2006)

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