Recent reports of conjunctivitis following human infection with H7 influenza viruses prompted us to conduct this study to better understand the ability of influenza viruses to use the eye as a portal of entry. Identification of influenza virus subtypes that readily infect ocular tissue may help us to better predict the risk of ocular infection and disease following exposure to influenza virus. In this study, we established mammalian in vivo, ex vivo, and in vitro models to assess the ability of influenza viruses of multiple subtypes to mount a productive infection following ocular inoculation and further identified corneal epithelial cells as a likely target for influenza virus replication. We found that, unlike human influenza viruses, specific avian influenza viruses within the H5 and H7 subtypes could successfully mount a systemic and lethal infection in the mouse following ocular inoculation and replicate to high titers in ocular tissues and cells.
We identified the HPAI H7N7 virus NL/219 and the HPAI H5N1 viruses HK/483 and Thai/16 as capable of causing a lethal infection in mice when introduced by the ocular route. These viruses also resulted in the highest lung virus titers observed among all strains tested and, following ocular inoculation, replicated to the highest titer in ex vivo mouse corneal tissue and in vitro HCEpiCs. Interestingly, the mortality observed in mice following ocular inoculation was delayed 3 to 4 days p.i. compared with mortality following intranasal inoculation with a similar dose of virus (4
). This relative delay in time to death following ocular inoculation may, in part, be attributed to the additional time necessary for virus to travel from the eye to the lung and establish a productive and ultimately fatal respiratory infection. Replication-independent spread of virus inoculum from the eye to the lung has been implicated previously in an ocular murine RSV model (6
), and a similar phenomenon could be occurring here. It should be noted that previous studies have used slightly lower inoculum volumes for ocular inoculation (6
); however, a volume of 5 μl was the minimum volume possible to achieve the high virus challenge dose (106
or PFU) examined in this study. It is reasonable to speculate that influenza virus travels from the eye via the lacrimal and nasolacrimal ducts to the nose, trachea, and finally lung. This transport appears to be unidirectional as intranasal inoculation of mice with these viruses does not result in viral titers in the eye (data not shown). Future studies examining ocular virus travel to the respiratory tract are warranted.
Despite a higher incidence of conjunctivitis following human H7 influenza virus infection in comparison with other virus subtypes, we found that HPAI H5N1 viruses replicated to substantial titers in ocular tissue ex vivo and HCEpiCs in vitro. However, upon in vivo ocular inoculation, H7N7 and H7N3 viruses were detected more frequently and at higher titer in the eye than H5N1 viruses, which were more frequently detected in respiratory tract tissues. Two additional HPAI H5N1 viruses that did not result in severe disease in the mouse model, VN/1203 and HK/486, replicated to titers comparable to those of other HPAI H5N1 viruses in HCEpiCs (data not shown) and may in part be due to the higher density of α2-3-linked SAs on the surface of the HCEpiCs compared with mouse corneas, as shown in Fig. . Not all H7 viruses replicated efficiently in ocular cells, and in the case of the LPAI NY/107 virus, this may be due, at least in part, to an increase in α2-6 SA binding in comparison to the classic avian-binding preference for α2-3-linked SAs observed with NL/219 or NL/230 virus (3
). While the precise molecular determinants that are responsible for this apparent ocular tropism have yet to be elucidated, this work reveals that highly pathogenic viruses of multiple subtypes are capable of replicating in ocular cells in vitro.
The human H1N1 1918 virus, which is highly lethal in mice when administered intranasally (42
), did not result in severe infection of mice when administered by the ocular route. Moreover, human influenza viruses of the H3N2 subtype were not detected in the eyes of mice following ocular inoculation and did not replicate to significant titers in vitro or ex vivo ocular cultures. While we did not observe efficient replication of these human viruses in vivo or in vitro, ocular symptoms occur sporadically following influenza infection with seasonal viruses (37
). Seasonal influenza vaccination has been associated rarely with the onset of oculo-respiratory syndrome in individuals with no prior history of ocular disease, and there have been sporadic reports of individuals rejecting corneal transplants following vaccination (12
). These adverse effects demonstrate the importance of studying the ocular environment in the context of both avian and human influenza virus infection and vaccination.
The presence of α2-3-linked SAs on the mouse and human cornea suggests that avian influenza viruses with this binding specificity would be well suited to infect this tissue and cause ocular disease. However, it appears that the ability of influenza viruses to bind to or replicate in ocular tissue cannot be explained by SA binding specificity alone. Both avian and human FITC-labeled viruses were capable of binding to ocular tissue regardless of SA binding preference. While the H5N1, H7N7, and H7N3 viruses included in this study all exhibit binding to α2-3-linked SAs, only those viruses within the H7 subtype replicated to substantial titer in the mouse eye in vivo, suggesting that an α2-3 SA binding preference alone is not sufficient to mount a productive ocular infection (3
). Furthermore, neither the reconstructed H1N1 1918 virus SC18 (α2-6 binding) nor the 1918 virus possessing the “avianized” HA (AV18; α2-3 binding) replicated in the eye, further indicating that properties other than SA binding preference contribute to the ability of selected influenza viruses to replicate in ocular tissue (45
). As we observed heightened α2-6 SA expression on the mouse corneal epithelium compared to HCEpiCs, subsequent study investigating the binding of these viruses on human tissue is warranted. Additionally, detailed characterization of the SAs present on the corneal epithelial sheet of the human eye is needed to better understand and identify receptors that are preferentially bound by influenza viruses. Recent studies examining the structural topology of surface glycans in the upper respiratory tract of humans has revealed greater complexity in the binding of influenza virus HA to α2-3 and α2-6 SAs than previously understood (10
); a similar analysis of ocular tissue may shed light on the precise composition of SAs on this surface and any correlation with preferential binding of influenza viruses, such as those within the H7 subtype, that are most frequently associated with conjunctivitis. A better understanding of the H7 and H5 subtype HA may further help elucidate why viruses within these subtypes, but not H3 or H1 viruses, were capable of replicating to high titer in the mouse lung following ocular inoculation; the lack of morbidity or mortality observed with either SC18 or AV18 viruses would suggest that, similar to replication within ocular tissue, properties in addition to SA binding preference are involved.
Similar to ocular models for RSV and herpes simplex virus (6
), we lightly scarified the mouse cornea prior to virus inoculation to reflect eye abrasions which have been reported to accompany ocular infection with avian strains in some studies. Multiple cases of human conjunctivitis have resulted from poultry workers involved in the culling of birds during avian influenza outbreaks, or others with exposure to poultry, reporting possible eye abrasions prior to onset of illness (21
). However, similar levels of virus replication and disease outcome were obtained in mice when influenza virus was administered by the ocular route in the absence of corneal scarification (data not shown), indicating that corneal scarification may not be required for avian influenza virus replication in the eye and subsequent spread to the respiratory tract. Unlike other ocular models of virus infection (6
), mice inoculated with influenza virus by the ocular route did not exhibit corneal disease, as measured by dissecting biomicroscope, with the exception of one NL/219-infected mouse that displayed moderate corneal opacity.
The observation of higher titers of replicating virus in mouse corneal epithelial sheets as compared with excised mouse corneas, in addition to the high titers observed following infection of HCEpiCs, suggests that the epithelial sheet is the primary site of virus replication in the cornea. However, SAs are also present on human conjunctiva, and H7N7 influenza viruses have been shown to bind to human conjunctiva epithelium in vitro (27
). With regard to the H7 viruses NL/230 and Can/504, which replicated to high titer in mouse eyes but did not replicate to significant titer in excised mouse corneas or corneal epithelial sheets, the conjunctiva may represent an additional in vivo site of virus replication which is absent in our ex vivo system. Future studies examining the role of this tissue in ocular infection are warranted. Definition of the role of ocular mucins following influenza virus infection could additionally allow for a more complete understanding of influenza virus infection of the ocular area.
While the majority of avian influenza virus infections in humans currently result from direct contact with infected or dead poultry, it is still unclear in some situations exactly how the virus travels to the respiratory tract (1
). Several H7 virus-infected individuals, including those from both the H7N7 outbreak in The Netherlands in 2003 and the H7N3 outbreak in Canada in 2004, reported both conjunctivitis and influenza-like respiratory illness following ocular exposure to virus (20
). Our findings of avian influenza viruses using the eye as a portal of entry to mount a productive and lethal infection in the mouse suggest that ocular exposure to some HPAI viruses could potentially result in the development of respiratory symptoms, similar to what was observed with these cases. In this study, H7 influenza viruses were most frequently associated with ocular disease, but avian viruses of the H5N1 subtype were also capable of using the eye as a portal of entry to initiate a productive infection. These results underscore the importance of wearing eye protection during possible exposure to avian influenza viruses of multiple subtypes (7