Methods S1 Supplementary methods. (DOCX) Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets While influenza viruses are a common respiratory pathogen, sporadic reports of conjunctivitis following human infection demonstrates the ability of this virus to cause disease outside of the respiratory tract. The ocular surface represents both a potential site of virus replication and a portal of entry for establishment of a respiratory infection. However, the properties which govern ocular tropism of influenza viruses, the mechanisms of virus spread from ocular to respiratory tissue, and the potential differences in respiratory disease initiated from different exposure routes are poorly understood. Here, we established a ferret model of ocular inoculation to explore the development of virus pathogenicity and transmissibility following influenza virus exposure by the ocular route. We found that multiple subtypes of human and avian influenza viruses mounted a productive virus infection in the upper respiratory tract of ferrets following ocular inoculation, and were additionally detected in ocular tissue during the acute phase of infection. H5N1 viruses maintained their ability for systemic spread and lethal infection following inoculation by the ocular route. Replication-independent deposition of virus inoculum from ocular to respiratory tissue was limited to the nares and upper trachea, unlike traditional intranasal inoculation which results in virus deposition in both upper and lower respiratory tract tissues. Despite high titers of replicating transmissible seasonal viruses in the upper respiratory tract of ferrets inoculated by the ocular route, virus transmissibility to naïve contacts by respiratory droplets was reduced following ocular inoculation. These data improve our understanding of the mechanisms of virus spread following ocular exposure and highlight differences in the establishment of respiratory disease and virus transmissibility following use of different inoculation volumes and routes. Despite reports of conjunctivitis following infection with numerous respiratory pathogens (including influenza, adenovirus, respiratory syncytial virus, and others), research investigating the role of ocular infection in virus pathogenicity and transmissibility has been underrepresented [1] [2] [3] [4] . Influenza virus represents a highly transmissible respiratory pathogen, resulting in .200,000 hospitalizations in the United States annually [5] . While ocular disease is generally rare following influenza virus infection in humans, viruses within the H7 subtype have demonstrated an apparent ocular tropism, with the majority of human infections with H7 influenza viruses associated with conjunctivitis [6] . Moreover, ocular complications have been sporadically documented following seasonal, 2009 H1N1 pandemic, and avian H5N1 virus infections in humans [7] [8] [9] [10] [11] [12] [13] .