Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets

Abstract

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.

Influenza Virus Infectivity and Virulence following Ocular-Only Aerosol Inoculation of Ferrets

“…However, it does not appear that virus replication in ocular tissue is necessary for subsequent respiratory infection, because not all productively infected ferrets had detectable virus in ocular samples (Tables 1 and 2). As in other models of ocular inoculation with influenza viruses (12,24), 100% infectivity of ferrets following OA inoculation was not observed, likely due to low presented doses, ferret-to-ferret physiological differences, or differences in the ocular surface area exposed during OA inoculation. The inclusion of an impinger sampler to measure the presence of aerosolized virus in the secondary chamber represents the most direct and comprehensive method possible to measure potential respiratory exposure during ocular-only inoculation; the absence of aerosolized virus outside the goggles during the inoculation and the lack of detection of virus in conjunctival wash specimens on day 1 p.i.…”

“…route suggest a potential mechanism for the reduced frequency of virus transmission observed in these studies (26). Accordingly, a decreased incidence of sneezing and nasal discharge was detected among ferrets inoculated by the ocular route (either with a liquid inoculum or by OA inoculation) (12), which could contribute to reduced transmissibility. Further study is needed to better understand the dissemination of virus from ocular tissues and the contribution this may make to virus transmission.…”

“…Several mammalian models that utilize an aerosolized virus inoculum have been established to address this need but have been restricted to inhalation exposure (3). Ferrets are used for the study of numerous viral pathogens (21,22) and were chosen to model ocular-only aerosol exposure because their physiology and distribution of cellular receptors in both respiratory and ocular tissues closely match those of humans (12,21). Therefore, while influenza virus was used in this study, the OA inoculation method presented here could easily be adapted for parallel studies with other pathogens that pose a similar threat to human health.…”

“…l Two additional ferrets received ocular doses of 68 and 118 PFU but did not shed virus in NW specimens and did not seroconvert; they are excluded from this analysis. (NW), conjunctival wash (CW), and rectal swab (RS) samples (12). All samples were titrated in MDCK cells by standard plaque assay as described previously (10).…”

Interaction of Influenza A/H1N1pdm Virus with Human Neuronal and Ocular Cells

“…Although a recent study showed H5N1 virus infection of ciliary process epithelial cells in ferrets, the permissiveness of human uveal cells to influenza virus infection and sialic-acid receptor expression patterns remain in question [2,43]. By analyzing the permissiveness to influenza A/H1N1pdm virus and sialic-acid receptor expression patterns of this panel of human neuronal and ocular cells in vitro, this study contributes to knowledge concerning neurological and ocular complications of influenza and possibly other viruses.…”