To determine if ocular exposure to influenza virus results in a transmissible respiratory infection, we inoculated ferrets by the Figure 2 . Comparison of influenza virus recovery in conjunctival wash samples 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 conjunctival washes (CW) collected on indicated days following serial titration in eggs; endpoint titers are expressed as mean log 10 EID 50 /ml plus standard deviation (left y-axis and bars). Relative viral RNA copy number in conjunctival washes was determined by real-time PCR using a universal M1 primer and extrapolated using a standard curve based on samples of known virus (right y-axis and lines). The limit of virus detection was 10 1.5 EID 50 /ml. {, no ferrets survived until day 9 p.i. R-squared values are shown for those viruses where a statistically significant (p,0.05) correlation between viral titer and viral RNA copy number exists. NS, not significant. doi:10.1371/journal.ppat.1002569.g002 PLoS Pathogens | www.plospathogens.org ocular route with selected influenza viruses known to transmit following traditional i.n. inoculation to naïve contacts in the presence of direct contact or by respiratory droplets ( Figure 5 ). Transmission was assessed by the detection of virus in NW samples and seroconversion of contact ferrets. To assess virus transmission in the presence of direct contact, ferrets were inoculated by the ocular route with the H7N2 virus NY/107 or the H7N7 virus NL/ 230, both viruses which transmit efficiently by this route following i.n. inoculation in ferrets [27] . Twenty-four hours later, a naïve ferret was placed in the same cage as each inoculated ferret to assess transmission. Both NY/107 and NL/230 viruses replicated efficiently in the inoculated ferrets following ocular inoculation as expected, and spread to 2/3 and 3/3 contact ferrets by day 7 postcontact (p.c.), respectively ( Figure 5A ). To assess virus transmissibility by respiratory droplets in the absence of direct contact, ferrets were inoculated by the ocular route with the H1N1 virus Brisbane or the H3N2 virus Panama, both viruses which transmit efficiently by this route following traditional i.n. inoculation [28, 30] . Twenty-four hours following i.o. inoculation, a naïve ferret was placed in an adjacent cage with modified side walls, so that air exchange was permitted between inoculated and contact ferrets in the absence of direct or indirect contact. Unlike the efficient transmission observed with these viruses following traditional i.n. inoculation, ferrets inoculated by the ocular route with either Brisbane or Panama only transmitted virus by respiratory droplets to 1/3 contact ferrets ( Figure 5B ). Virus was not detected in CW or RS samples from the infected Brisbane contact ferret; the infected Panama contact ferret had a peak CW titer of 10 2.75 EID 50 day 5 p.c. and peak RS titer of 10 2.5 EID 50 day 7 p.c. While RD contacts with detectable virus in NW samples seroconverted to homologous virus at the end of the experimental period, contact ferrets which did not have detectable virus in NW samples did not exhibit seroconversion (data not shown). Ferrets inoculated intranasally with 10 6 EID 50 of Brisbane virus in a reduced 100 ml volume and tested for their ability to transmit virus by respiratory droplets exhibited a similar pattern of virus transmissibility as ferrets inoculated by the ocular route, with virus shedding and seroconversion detected in only 1/3 contact ferrets (data not shown). These findings suggest that despite high titers of virus in NW samples, the respiratory infection resulting from inoculation of ferrets with a reduced volume, by either ocular or intranasal inoculation routes, is distinct from that following traditional i.n. inoculation, characterized by a diminished incidence of sneezing and nasal discharge and resulting in reduced transmission of virus by respiratory droplets.