Influenza A and B Virus Attachment to Respiratory Tract 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.

U nderstanding is limited about factors determining the ability of infl uenza viruses to cross the species barrier and persist in a new host population (1,2). In marine mammals, several subtypes of avian infl uenza A virus have caused epidemics in harbor seals (Phoca vitulina) (3)(4)(5)(6). Also, human infl uenza B virus has been detected in harbor seals (7). These observations indicate the ability of both viruses to cross the species barrier and persist in harbor seals. In other marine mammal species, outbreaks of avian infl uenza A virus or infection with human infl uenza B virus have not been reported.
Attachment of infl uenza virus to tissues in the respiratory tract is a major determinant of host susceptibility to infection, effi ciency of transmission, and pathogenicity and has been studied only to a limited degree (8,9). Attachment is determined largely by the specifi city with which infl uenza virus attaches to sialosaccharide receptors on the host cell surface. In general, human infl uenza viruses prefer sialosaccharides in which sialic acid is linked to galactose by an α-2,3 linkage (SA-α-2,3-Gal), and avian infl uenza viruses prefer those with an α-2,6 linkage (SAα-2,6-Gal) (10).
To understand differences in these properties between harbor seals and other marine mammals, we determined patterns of attachment for infl uenza virus strains known to have infected the respiratory tract of harbor seals, gray seals (Halichoerus grypus), harbor porpoises (Phocoena phocoena), and bottlenose dolphins (Tursiops truncatus). We chose gray seals, porpoises, and dolphins because their ranges overlap those of harbor seals and they are commonly kept in captivity.

The Study
We determined patterns of attachment to respiratory tract tissues of 4 sympatric marine mammal species for several infl uenza viruses. Avian infl uenza 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 infl uenza 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. Infl uenza 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. All viruses were isolated as described (11)(12)(13).
We obtained respiratory tract specimens from marine mammals from archives of paraffi n-embedded tissues. Trachea and lung (including bronchus, bronchiole, and pulmonary alveoli) from 3 animals per species were examined.
Attachment of infl uenza 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. Staining was scored as the percentage of cells in a section showing virus attachment. We also evaluated virus attachment to submucosal glands.
Results of attachment differed between avian infl uenza A viruses, human infl uenza A viruses, and human infl uenza B viruses. First, attachment of avian infl uenza 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. There were a few exceptions for virus attachment (Table). The source of avian infl uenza viruses (mallard or harbor seals) did not have a consistent effect on virus attachment in respiratory tract epithelium of any evaluated species (Table). Second, attachment of human infl uenza 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 infl uenza virus A(H1N1) pdm09 and seasonal subtype (H3N2) virus to respiratory tract tissues of cetaceans (Table).
Third, attachment of infl uenza 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).

Conclusions
Attachment of avian infl uenza A viruses to the respiratory tract was generally consistent with reports, or lack thereof, of avian infl uenza in these 4 marine mammal species. Moderate attachment of avian infl uenza A viruses to the trachea and bronchi of harbor seals suggests high susceptibility to and effi cient transmission of these viruses. This fi nding is consistent with reported outbreaks of avian infl uenza in harbor seals (4)(5)(6). Scarce attachment of avian infl uenza viruses to bronchioles and alveoli of harbor seals is consistent with low pathogenicity of these viruses for harbor seals during experimental infection (4)(5)(6).
Attachment of avian infl uenza A virus to the respiratory tract in gray seals strongly resembles attachment in harbor seals. However, infection or outbreaks of avian infl uenza A virus in gray seals have not been reported, probably because virus attachment is required but is not suffi cient for infection. Lack of attachment of avian infl uenza A viruses to trachea and bronchi of harbor porpoises and bottlenose dolphins suggests low susceptibility and ineffi cient transmission. This fi nding is consistent with lack of reported avian infl uenza A virus infections in these species (14).
Absence or scarcity of attachment of human infl uenza 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-α-2,6-Gal (15). This fi nding suggests low susceptibility to infection and can explain the lack of reported human infl uenza 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 infl uenza B virus to the respiratory tract of the 4 marine mammal species resembled that of the avian infl uenza A viruses. Moderate attachment of infl uenza B virus to the respiratory tract of seals suggests high susceptibility and effi cient transmission. This fi nding is consistent with isolation of infl uenza B virus from a harbor seal and serologic evidence of infl uenza B virus infection in gray seals (7). Lack of attachment of infl uenza B virus to trachea and bronchi of cetaceans is consistent with absence of reported infl uenza B virus infections in these species.
Source of virus strain had little effect on its attachment. In general, there was high similarity of attachment of avian infl uenza A viruses from harbor seals and mallards. These fi ndings suggest that avian infl uenza viruses do not require a different pattern of attachment to infect and transmit effi ciently among harbor seals and that harbor seals might be susceptible to a wider range of avian infl uenza viruses than reported.
In conclusion, we report extensive diversity in the pattern of attachment of infl uenza viruses to the respiratory tract of marine mammals, which was determined by virus strain and host species involved. Our results correspond to fi eld observations of infl uenza in marine mammals, i.e., outbreaks of avian infl uenza A virus and human infl uenza B virus infection in harbor seals (4,5) and lack of evidence of human infl uenza A virus infection in marine mammals. These results suggest that, as in humans (11,15), attachment of infl uenza virus to the proximal part of the respiratory tract, which depends largely on appropriate sialic acid moieties, is critical for susceptibility and effi cient transmission of infl uenza viruses in marine mammals.