we determined patterns of attachment to respiratory tract tissues of 4 sympatric marine mammal species for several influenza viruses . avian influenza a virus subtypes h7n7 ( a / seal / massachusetts/1/80 ) and h4n5 ( a / seal / ma/47/83 ) were chosen because they had caused outbreaks in harbor seals ( 4,5 ) . an influenza b virus strain ( b / seal / netherlands/1/99 ) was chosen because it had been isolated from a harbor seal ( 7 ) . for each of these 3 viruses , we also included a closely related strain from the putative donor host species ( h7n7 a / mallard / sweden/100/02 , h4n5 a / mallard / netherlands/13/2008 , and b / harbin/7/94 , respectively ) to determine whether adaptation to the new host species was associated with a change in attachment . influenza virus a(h1n1)pdm09 ( a / netherlands/164/09 ) and seasonal subtype ( h3n2 ) virus ( a / netherlands/213/03 ) were chosen because they circulate endemically in humans and might have contact with captive marine mammals through their caretakers . we obtained respiratory tract specimens from marine mammals from archives of paraffin - embedded tissues . trachea and lung ( including bronchus , bronchiole , and pulmonary alveoli ) from 3 animals per species were examined . attachment of influenza virus to tissues was visualized by histochemical analysis as described ( 13 ) . a positive result by light microscopy was granular to diffuse red staining on the apical surface of epithelial cells in trachea , bronchi , and bronchioles and on alveolar cells . results of attachment differed between avian influenza a viruses , human influenza a viruses , and human influenza b viruses . first , attachment of avian influenza a viruses to tracheal and bronchial epithelium was moderate in seals ( harbor seal and gray seal ) and absent in cetaceans ( harbor porpoise and bottlenose dolphin ) ( figure ) . attachment to bronchiolar epithelium was moderate in seals and scarce in cetaceans , and attachment to alveolar epithelium was scarce in all 4 species . the source of avian influenza viruses ( mallard or harbor seals ) did not have a consistent effect on virus attachment in respiratory tract epithelium of any evaluated species ( table ) . attachment of 2 human influenza viruses and 1 avian influenza virus to trachea and bronchiole of harbor seal ( phoca vitulina ) and harbor porpoise ( phocoena phocoena ) . red staining indicates virus on the surface of epithelial cells ( histochemical staining counterstained with hematoxylin ; original magnification 100 ) . * mean abundance of cells to which virus attached was scored as follows:+ , moderate ( 10%50% cells positive ) ;  , scarce ( < 10% cells positive ) ;  , negative ( no attachment ) ; + + , abundant ( > 50% cells positive ) . cil , ciliated epithelial cell ; nd , not determined ; i , type i pneumocytes ; ii , type ii pneumocytes . second , attachment of human influenza a viruses to tracheal and bronchial epithelium was absent in seals and scarce in cetaceans . attachment to bronchiolar and alveolar epithelium was absent or scarce in seals and moderate to abundant in cetaceans ( table , figure ) . we detected few differences between attachment of influenza virus a(h1n1)pdm09 and seasonal subtype ( h3n2 ) virus to respiratory tract tissues of cetaceans ( table ) . third , attachment of influenza b viruses to respiratory tract epithelium at all levels was scarce to moderate in seals . attachment was negative for tracheal and bronchial epithelium , scarce for bronchiolar epithelium , and moderate for alveolar epithelium in cetaceans ( table , figure ) . attachment of avian influenza a viruses to the respiratory tract was generally consistent with reports , or lack thereof , of avian influenza in these 4 marine mammal species . moderate attachment of avian influenza a viruses to the trachea and bronchi of harbor seals suggests high susceptibility to and efficient transmission of these viruses . this finding is consistent with reported outbreaks of avian influenza in harbor seals ( 46 ) . scarce attachment of avian influenza viruses to bronchioles and alveoli of harbor seals is consistent with low pathogenicity of these viruses for harbor seals during experimental infection ( 46 ) . attachment of avian influenza a virus to the respiratory tract in gray seals strongly resembles attachment in harbor seals . however , infection or outbreaks of avian influenza a virus in gray seals have not been reported , probably because virus attachment is required but is not sufficient for infection . lack of attachment of avian influenza a viruses to trachea and bronchi of harbor porpoises and bottlenose dolphins suggests low susceptibility and inefficient transmission . this finding is consistent with lack of reported avian influenza a virus infections in these species ( 14 ) . absence or scarcity of attachment of human influenza a viruses to trachea and bronchi of any of the marine mammal species contrasts with that of humans ( 13 ) , in whom trachea and bronchi mainly express sa--2,6-gal ( 15 ) . this finding suggests low susceptibility to infection and can explain the lack of reported human influenza a virus infections in these 4 marine mammal species ( 14 ) , even though they are often kept in captivity and are therefore at risk for infection from humans . attachment of influenza b virus to the respiratory tract of the 4 marine mammal species resembled that of the avian influenza a viruses . moderate attachment of influenza b virus to the respiratory tract of seals suggests high susceptibility and efficient transmission . this finding is consistent with isolation of influenza b virus from a harbor seal and serologic evidence of influenza b virus infection in gray seals ( 7 ) . lack of attachment of influenza b virus to trachea and bronchi of cetaceans is consistent with absence of reported influenza b virus infections in these species . in general , there was high similarity of attachment of avian influenza a viruses from harbor seals and mallards . these findings suggest that avian influenza viruses do not require a different pattern of attachment to infect and transmit efficiently among harbor seals and that harbor seals might be susceptible to a wider range of avian influenza viruses than reported . in conclusion , we report extensive diversity in the pattern of attachment of influenza viruses to the respiratory tract of marine mammals , which was determined by virus strain and host species involved . our results correspond to field observations of influenza in marine mammals , i.e. , outbreaks of avian influenza a virus and human influenza b virus infection in harbor seals ( 4,5 ) and lack of evidence of human influenza a virus infection in marine mammals . these results suggest that , as in humans ( 11,15 ) , attachment of influenza virus to the proximal part of the respiratory tract , which depends largely on appropriate sialic acid moieties , is critical for susceptibility and efficient transmission of influenza viruses in marine mammals .
patterns of virus attachment to the respiratory tract of 4 marine mammal species were determined for avian and human influenza viruses . 
 attachment of avian influenza a viruses ( h4n5 ) and ( h7n7 ) and human influenza b viruses to trachea and bronchi of harbor seals is consistent with reported influenza outbreaks in this species .