on april 21 , 2008 , three whooper swans ( 2 adults and 1 juvenile ) were found dead at lake towada , akita prefecture , japan ( figure 1 ) .
it was taken to the wildlife protection center in akita but had to be euthanized that day in moribund status .
homogenates from the tracheas , cloacas , and internal organs of 3 swans were pooled and inoculated into embryonated chicken eggs for virus isolation .
map of japan and nearby countries , with enlargement of the northern part of the country ( inset ) showing location of lake towada .
agents were confirmed to be type a influenza viruses by a commercial rapid antigen assay kit and were excluded from being newcastle disease virus by the hemagglutination inhibition test with newcastle - specific antiserum .
after those tests conducted at the animal hygiene service center , viruses were brought to the national institute of animal health , tsukuba , japan , for further analysis .
the viruses were subtyped as h5n1 with a panel of antiserum , and 1 yielded from cloaca homogenates was designated as a / whooper swan / akita/1/2008 ( wsak08 ) and was further analyzed .
wsak08 was shown to be highly pathogenic to chickens by an intravenous administration of 10-fold diluted infectious allantoic fluid .
this result coincides with the sequence analysis of the hemagglutinin ( ha ) gene , showing that the ha protein possesses a series of basic amino acids ( pqrerrrkr ) at the cleavage site .
phylogenetic analysis of the ha1 region of the ha gene ( figure 2 ) showed that wsak08 belongs to clade 2.3.2 and is clearly distinguishable from the hpaivs previously isolated in japan in 2004 , a / chicken / yamaguchi/7/2004 ( clade 2.5 ) , and in 2007 , a / chicken / miyazaki / k11/2007 ( clade 2.2 ) . although sequence data were not found in genbank , a / common magpie / hong kong/5052/2007 reportedly resides in the same clade ( 9 ) .
antigenic analysis of wsaki08 with a panel of antiserum and monoclonal antibodies showed low reactivity against antibodies in the panel ( appendix table ) . a > 32-fold reduction from homologous titers of all hyperimmune serum used
postinfection duck serum against a / chicken / yamaguchi/7/2004 and a / chicken / miyazaki / k11/2007 did not react with wsak08 .
none of the monoclonal antibodies against ha protein of a / chicken / yamaguchi/7/2004 reacted with wsak08 .
thus , wsak08 is genetically and antigenically distinguishable from the hpaivs that caused previous outbreaks in japan .
phylogenetic tree constructed based on the hemagglutinin ( ha ) 1 region ( 966 bp ) of the ha gene of the highly pathogenic avian influenza viruses ( h5n1 ) .
clade designation follows the criteria proposed by the world health organization / world organisation for animal health / food and agriculture organization h5n1 evolution working group ( 8) .
representative strains of the previous highly pathogenic avian influenza outbreaks in japan are in boldface .
ab436731ab436738 ) showed that it does not contain amino acid substitutions conferring resistance to adamantane or neuraminidase inhibitors . unlike many isolates related to qinghai lake strains that have spread worldwide
the neuraminidase protein has a 20-aa deletion at aa 49 to 68 in the stalk region .
nonstructural protein 1 has a 5-aa deletion at aa 80 to 84 , commonly observed in currently circulating hpaivs ( h5n1 ) in southeastern asia .
whooper swans breed in northern eurasia and winter in europe and eastern asia i.e. , china , the korean peninsula , and japan . in japan
, 35,00038,000 whooper swans spend every winter primarily in hokkaido , tohoku , and the hokuriku area ( 10 ) . in the lake towada area , 300 whooper swans arrive beginning in late october ; they leave the area between late march and late april ( 11 ) . in late march , summer birds begin to arrive . according to results of satellite tracking of 8 swans ( 12 ) , as well as the results of banding studies since 1961 ( 13 ) ,
whooper swans that winter in japan migrate from the northern end of honshu island to eastern hokkaido , by means of sakhalin , and reach eastern siberia , where they breed . to our knowledge , there have been no reports of whooper swans that winter on the eurasian continent migrating north through japan . in light of the migratory route mentioned above , the whooper swans found dead at lake towada were most likely recently infected with hpaiv ( h5n1 ) in japan .
it is unlikely that the swans were infected before they flew to japan in autumn , maintained the virus within the flock , and then suddenly developed the disease after no apparent infections for several months .
although the susceptibility of a certain species of birds to a subtype h5n1 virus may be different depending on the virus strain ( 14 ) , whooper swans as well as mute swans have been considered to be susceptible species to hpaiv ( h5n1 ) , as they showed a fulminant course of disease at the outbreak in germany in 2006 ( 7 ) .
the possibility that the swans were infected by domestic fowl is low because there has been no report of hpai among domestic fowl in japan since the beginning of 2008 .
one possible explanation is that other wild birds brought the virus from outside the country .
although it is not known whether any birds wintering on the continent migrate north through japan , passage visitor birds such as wader birds migrate from south to north through japan in spring ; summer birds , e.g. , egrets , swallows , songbirds , and some raptors , come to japan from the south in spring for breeding .
also , the possibility of anthropogenic introduction of virus , such as by inappropriate importation of birds , meats , or materials , can not be excluded . in conclusion
, genetic analysis demonstrates that the virus that killed the 4 swans in japan in 2008 is genetically distinguishable from the strains that caused previous poultry outbreaks in japan , ruling out a possibility of resurgence of previously introduced hpaiv in japan . after the incident we describe
, 2 other whooper swan cases of hpaiv ( h5n1 ) infection were confirmed in eastern hokkaido in early may .
possible involvement of wild birds in the introduction of the virus to japan requires further scrutiny .
antigenic analysis of whooper swan / akita/1/2008 highly pathogenic avian influenza virus ( h5n1 ) versus related isolates *
 on april 21 , 2008 , four whooper swans were found dead at lake towada , akita prefecture , japan . 
 highly pathogenic avian influenza virus of the h5n1 subtype was isolated from specimens of the affected birds . 
 the hemagglutinin ( ha ) gene of the isolate belongs to clade 2.3.2 in the ha phylogenetic tree . 