The detection of high viral titers in NW samples as early as day 1 p.i. following i.o. inoculation suggests replication-independent Figure 1 . Comparison of mean titers of influenza viruses recovered from nasal wash following ocular inoculation of ferrets. Ferrets were inoculated by the ocular route with 10 6 EID 50 /ml of each virus shown. Viral titers were measured in nasal washes collected on indicated days following serial titration in eggs; endpoint titers are expressed as mean log 10 EID 50 /ml plus standard deviation. The limit of virus detection was 10 1.5 EID 50 /ml. {, ferrets did not survive to day 9 p.i. doi:10.1371/journal.ppat.1002569.g001 spread of virus from the eye to the respiratory tract ( Figure 1 ); this has been similarly hypothesized in previous studies, but has yet to be proven experimentally [20, 33, 34] . Reduced viral titers in the lungs of ferrets on day 3 p.i. following ocular compared with intranasal administration further indicates differential patterns of virus spread following inoculation (Table 3) . To visualize the deposition of virus immediately following different routes of inoculation, we labeled NL/219 virus with an AF680 fluorescent tag (NL/219-FL) and inoculated ferrets with equal quantities of NL/219-FL virus by the ocular (100 ml total volume) or intranasal (1 ml total volume diluted in PBS) route ( Figure 4 ). Ferrets were euthanized 15 minutes following virus inoculation for ex vivo imaging. In ferrets inoculated by the traditional intranasal route, the majority of virus was deposited in the nasal turbinates and lungs, consistent with a previous study demonstrating virus dissemination throughout upper and lower respiratory tract tissue following this route of inoculation [32] . In contrast, virus deposition in ferrets inoculated by the ocular route (right side only) was primarily localized in the nasal turbinates and right conjunctiva. Lower relative quantities of virus inoculum were present in the upper trachea and esophagus following either intranasal or ocular inoculation. These findings demonstrate that, following i.o. inoculation in ferrets, influenza virus rapidly spreads to the nasal turbinates and upper trachea in a replicationindependent manner, but in contrast to i.n. inoculation, does not immediately deposit in peripheral lung tissue. Furthermore, initial deposition of virus inoculum following ocular inoculation occurs not on the corneal surface of the eye but is rather concentrated in the surrounding conjunctival tissue.