we analyzed the amino acid sequence of the receptor binding site of ha from the isolate a / jiangxi - donghu/346 - 1/2013 ( h10-jd346 ; global initiative on sharing avian influenza data [ gisaid , http://www.gisaid.org ] accession no .
epi530526 ) from the first patient infected by influenza a(h10n8 ) virus . in addition , several human and avian influenza viruses ( sequences from gisaid or the national center for biotechnology information website ) and a recent harbor seal isolate ( 5 ) were compared with h10-jd346 ( table ) .
we observed that residues involved in receptor binding for h10 subtype influenza viruses suggest avian - like receptor specificity .
however , we identified 2 amino acids in avian and human h10 , t135 and s186 , that are common in circulating human influenza viruses and were associated with changes in receptor binding in other avian influenza a virus subtypes ( 6,7 ) . in accordance with this finding , vachieri et al
. found substantial levels of binding of an avian h10 ha to sa2,6 that retained the ability to interact with sa2,3 ( 8) .
* residues found in human h1 or h3 and in h10 hemagglutinin but not in other avian hemagglutinin sequences are shown in bold . given the role of receptor binding specificity of emerging influenza viruses , we analyzed the interaction of ha of the human h10-jd346 influenza a(h10n8 ) virus isolate in comparison with that of an avian h10n7 subtype virus .
first , we used a solid - phase binding assay ( 9,10 ) and the following biotinylated glycans conjugated with a polyacrylamide ( paa ) support ( provided by the consortium of functional glycomics [ cfg ] ) : neu5ac2,6gal14glcnac-paa ( 6 sln - paa ) ; neu5ac26(gal14glcnac13)2-paa ( 6sdi - ln - paa ) ; neu5ac2,3gal14glcnac-paa ( 3 sln - paa ) ; neu5ac23(gal14glcnac13)2-paa ( 3sdi - ln - paa ) ; and neu5ac23(gal14glcnac-sp)3-paa ( 3stri - ln - paa ) .
we also analyzed recombinant hexahistidine - tagged has ( 11 ) from h10-jd346 , an avian h10n7 subtype strain from north america ( a / mallard / interior alaska/10bm01929/2010 ; h10-mallard ) , a human h3n2 subtype seasonal influenza a virus ( a / panama/2007/1999 ; h3-p99 ) , and an h5n1 subtype avian influenza virus from a fatal human case ( a / vietnam/1203/2004 ; h5-viet ) .
as expected , h3-p99 bound strongly to the sa2,6 tested , and h5 showed higher levels of binding to sa2,3 than to sa2,6 ( figure 1 , panel a ) .
when we analyzed h10-mallard and h10-jd346 , we found a similar binding profile , which is consistent with the presence of similar amino acids affecting the receptor binding specificity ( table ) .
although both h10 proteins had a prevalent avian - like binding profile , low levels of binding to sa2,6 were also observed .
a ) binding of recombinant hemagglutinins to glycans in a solid - phase binding assay .
( human ) , h5-viet ( avian origin isolated from a human case ) , and h10-mallard ( avian ) viruses were included in the analysis for comparison and as controls .
values at the top right of the dot plots indicate percentage of cells expressing matrix protein 2 ( m2 ) .
fsc , forward - scattered light ; fitc , fluorescein isothiocyanate .   to confirm this data , we used a flow cytometry  based assay and the same synthetic glycans ( 9,10 ) .
we infected mdck epithelial cells with h10-jd346 virus ( 6:2 re - assortant with the backbone of laboratory strain a / puerto rico/8/1934 [ pr8 ] , which was generated as described ) ( 9,10 ) ; h10-mallard ( wild - type ) ; human isolate h3-p99 ( wild - type ) ; and h5-viet 6:2 ( low pathogenicity reassortant with the backbone of pr8 ) ( 9,10 ) at a multiplicity of infection of 1 .
cells were harvested 24-h postinfection and incubated with antibody against matrix protein 2 ( e10 ) , which was detected by using an antibody against igg ( alexa 647 antibody ; invitrogen , carlsbad , ca , usa ) as a control of infection and with the sialyl - glycans ( detected with streptavidin  fluorescein isothiocyanate ; jackson laboratories , bar harbor , me , usa ) .
we determined the percentage of infected cells in each sample and gated the infected population to determine the sa binding profile ( figure 1 , panel b ) .
h3-p99 showed high levels of binding to sa2,6 and h5-viet bound more efficiently to sa2,3 than to sa2,6 , which is similar to observations with recombinant has in the solid - phase binding assay .
h10-mallard and h10-jd346 showed similar binding profiles with preferential binding for sa2,3 and binding to sa2,6 slightly higher than that for the negative control .
analysis of receptor binding of h10-jd346 and of h10-mallard with 2 independent assays indicated that the h10 subtype influenza virus interacts slightly with human - like receptors and maintains preferential binding to avian - like receptors .
consequently , these data suggest that h10 subtype influenza virus might have the ability to interact with the upper human respiratory tract , which is rich in sa2,6 ( 3 ) . to test this hypothesis , we precomplexed h3-p99 and h10-jd346 with primary antibody ( mouse anti - his tag ) and secondary fluorescent antibody , then incubated the complex with 2 human tracheal samples ( 12 ) . as expected ,
recombinant h10-jd346 ha also interacted with respiratory epithelia ( figure 2 ) , which suggested that the virus might be able to attach and replicate in the human upper respiratory tract .
however , the 6:2 reassortant virus h10-jd346 virus showed markedly decreased replication compared with that of an h3n2 subtype virus ( pr8 6:2 reassortant ) in a human lung epithelial cell line ( figure 3 ) .
interaction of hemagglutinin ( ha ) of h3-p99 ( panels b and e ) and h10-jd346 ( panels c , f , and g ) isolates of influenza a(h10n8 ) viruses with human trachea .
scale bars indicate 25 m .   replication of influenza a(h10n8 ) h10-jd346 virus in human epithelial cells .
a549 cells ( a human lung epithelial adenocarcinoma cell line ) were infected at a multiplicity of infection of 0.1 with the h10-jd346 virus ( 6:2 re - assortant with the backbone of pr8 ) and another 6:2 re - assortant virus expressing hemagglutinin and neuraminidase genes from a human influenza a(h3n2 ) virus ( a / wyoming/3/2003 ) .
cells were incubated at 37c in dulbecco minimal essential medium containing 0.3% bovine albumin ( mp biomedicals , solon , oh , usa ) and 1 g / ml of tolylsulfonyl phenylalanyl chloromethyl ketone  treated trypsin ( sigma , st .
supernatants were collected at selected time points , and viral titers on mdck cells were determined by using a standard plaque assay .
ha of novel influenza a(h10n8 ) virus interacts with sa2,3 and slightly with sa2,6 , at levels similar to that for an avian h10 subtype ha , and binds to cells in the human upper respiratory tract .
variations in the experimental settings and protocols ( e.g. , concentration of ha or glycans used ) might account for these dissimilarities .
an epidemic among seals caused by this virus subtype is currently ongoing in europe ( 5 ) .
a study by beare and webster showed that 50% of volunteers experimentally infected with influenza a(h10n7 ) virus shed virus ( 15 ) , which our data suggests might be caused by initial attachment to the upper respiratory tract .
immune responses were not detected in these volunteers , and mild , if any , symptoms developed , which indicated limited virus replication . the low incidence of h10 influenza virus indicates a limited pandemic potential of h10n7 and h10n8 viruses .
therefore , further changes in receptor binding , as well as acquisition of genomic segments from other avian influenza virus strains through co - infection , would be required to increase fitness and transmissibility in mammals .
isolate h10-jd346 amino acid sequence had a mixture of e and k in position 627 of basic polymerase protein 2 ; the k627 mutation is associated with mammal adaptation ( 1 ) .
this finding highlights the need for an efficient surveillance network to track and identify possible changes , as well as extensive research to identify them and understand their functional consequences .
 three cases of influenza a(h10n8 ) virus infection in humans have been reported ; 2 of these infected persons died . 
 characterization of the receptor binding pattern of h10 hemagglutinin from avian and human isolates showed that both interact weakly with human - like receptors and maintain strong affinity for avian - like receptors . 
The Study
Conclusions