Influenza virus infectivity and virulence following ocular-only aerosol inoculation of ferrets

Abstract

Respiratory pathogens have traditionally been studied by examining the exposure and infection of respiratory tract tissues. However, these studies typically overlook the role of ocular surfaces, which represent both a potential site of virus replication and a portal of entry for the establishment of a respiratory infection. To model transocular virus entry in a mammalian species, we established a novel inoculation method that delivers an aerosol inoculum exclusively to the ferret ocular surface. Using influenza virus as a representative respiratory pathogen, we found that both human and avian viruses mounted productive respiratory infections in ferrets following ocular-only aerosol inoculation, and we demonstrated that H5N1 virus can result in a fatal infection at doses below 10 PFU or with exposure times as short as 2 min. Ferrets inoculated by the ocular aerosol route with an avian (H7N7, H7N9) or human (H1N1, H3N2v) virus were capable of transmitting the virus to naïve animals in direct-contact or respiratory-droplet models, respectively. Our results reveal that ocular-only exposure to virus-containing aerosols constitutes a valid exposure route for a potentially fatal respiratory infection, even for viruses that do not demonstrate an ocular tropism, underscoring the public health implications of ocular exposure in clinical or occupational settings.

Importance: In the absence of eye protection, the human ocular surface remains vulnerable to infection with aerosolized respiratory viruses. In this study, we present a way to inoculate laboratory mammals that excludes respiratory exposure, infecting ferrets only by ocular exposure to influenza virus-containing aerosols. This study demonstrates that the use of respiratory protection alone does not fully protect against influenza virus exposure, infection, and severe disease.

FIG 1

Graphical representation of ocular aerosol exposure system. An influenza virus aerosol system used for inhalation exposure of ferrets was modified to provide ocular-only aerosol exposure to ferrets. (Inset) Close-up of the aerosol delivery goggles used in ferret experiments.

FIG 2

A/Thailand/16/04 viral titers in nasal wash specimens and tissues following OA inoculation. (A and B) Ferrets were inoculated by the OA route with a high (>100-PFU) (A) or low (<100-PFU) (B) dose of virus, and virus titers in nasal wash specimens were determined on the indicated days p.i. Each line represents an individual ferret receiving the indicated dose. The duration of exposure is given in parentheses in the key. (C) Box-and-whisker plots of titers in tissues collected from ferrets euthanized due to neurological symptoms on days 5 to 8 after inoculation with the Thai/16 virus. The mean numbers of ferrets with positive virus detection in each tissue are shown; the number of ferrets with positive virus detection out of the total number of ferrets sampled is given above each box-and-whisker plot. Nas Tur, nasal turbinates; BnOB, olfactory bulb; Conj, conjunctiva. Titers are expressed in PFU/g in all tissues except for Nas Tur, Eye, and Conj, in which titers are expressed in PFU/ml. Eye and conjunctiva samples include both left and right tissues. “Brain” includes both anterior and posterior segments. The limit of detection for all titrations was 10 PFU.

FIG 3

Titers of influenza viruses recovered from ferret nasal wash specimens following OA inoculation. Ferrets were inoculated by the OA route with the indicated viruses at the doses reported in Table 2. Viral titers were measured in nasal wash specimens collected on the indicated days p.i. Lines represent individual ferrets. The limit of detection was 10 PFU.

FIG 4

Transmissibilities of influenza viruses in ferrets following OA inoculation. Three ferrets were inoculated by the OA route with the indicated viruses at the doses reported in Table 2, and nasal wash specimens were collected from each ferret on the indicated days p.i. (solid bars). A naïve ferret was placed either in the same cage as each inoculated ferret (A) or in an adjacent cage with perforated side walls (B) at 24 h p.i., and nasal wash specimens were collected from each contact ferret on the indicated days p.c. (hatched bars) in order to assess virus transmission in the presence of direct contact or respiratory droplets, respectively. The limit of virus detection was 10 PFU.