Ocular tissue is not routinely titrated following i.n. inoculation of influenza virus in ferrets, making it difficult to elucidate if viral titers in ocular tissue are a function of i.o. inoculation or are detected regardless of the inoculation route. Therefore, we collected both left and right whole ferret eyes and all surrounding conjunctiva/eyelid for virus titration from ferrets inoculated by the intranasal or ocular route with NL/219, NL/230, or Brisbane viruses (Table 4) . Surprisingly, sporadic viral titers from both left and right eye and conjunctival tissue were detected following HPAI H7N7 virus infection by both i.n. (using either a 1 mL or 100 ml inoculation volume) or i.o. inoculation routes (Table 4) . While the magnitude of viral titers and viral RNA was generally similar between intranasal and ocular routes of inoculation, realtime RT-PCR detected CW-positive samples with a greater sensitivity compared with viral culture. Isolation of virus from ocular tissue may be a reflection of the ability of these HPAI viruses to spread to extra-pulmonary tissues post-inoculation as previously described [26] . However, virus was also detected in left and right conjunctival tissue following i.n. or i.o. inoculation of the H1N1 virus Brisbane, a virus which lacks a high capacity for systemic spread [28] . Comparable levels of viral RNA were isolated from CW samples from ferrets inoculated with Brisbane virus by either intranasal or ocular routes, although infectious virus was only detected in CW samples collected from the eyes of ferrets inoculated by the ocular route. To confirm that virus detected in the eye and conjunctiva was associated with tissue-specific virus replication, immunohistochemistry (IHC) was performed to visualize the presence of influenza A nucleoprotein (NP) in ferret ocular tissues. As shown in Figure 3 , influenza virus antigen was detected in epithelial cells from both the lacrimal glands in the conjunctiva and the ciliary processes in the eye collected day 3 p.i. from ferrets inoculated by the ocular route. These results indicate that the route of virus inoculation in ferrets can affect the extent of virus dissemination in respiratory tract tissue, but extra-pulmonary spread, notably to ocular tissue, is present regardless of the point of entry once an infection is established.