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. 2012;8(3):e1002569.
doi: 10.1371/journal.ppat.1002569. Epub 2012 Mar 1.

Influenza virus respiratory infection and transmission following ocular inoculation in ferrets

Jessica A Belser  1 , Kortney M Gustin, Taronna R Maines, Mary J Pantin-Jackwood, Jacqueline M Katz, Terrence M Tumpey
Affiliations

Influenza virus respiratory infection and transmission following ocular inoculation in ferrets

Jessica A Belser et al. PLoS Pathog. 2012.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
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 106 EID50/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 log10 EID50/ml plus standard deviation. The limit of virus detection was 101.5 EID50/ml. †, ferrets did not survive to day 9 p.i.
Figure 2
Figure 2. Comparison of influenza virus recovery in conjunctival wash samples following ocular inoculation of ferrets.
Ferrets were inoculated by the ocular route with 106 EID50/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 log10 EID50/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 101.5 EID50/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.
Figure 3
Figure 3. Photomicrographs of ferret tissue sections stained for the presence of influenza viral antigen following ocular inoculation.
Ferrets were inoculated by the ocular route with 106 EID50/ml of NL/230 or Brisbane virus, and tissues were collected day 3 p.i. for analysis. Viral antigen (staining in red) found in epithelial cells of lacrimal glands in the conjunctiva of a ferret inoculated with Brisbane virus (A) and epithelial cells of the ciliary processes in the eye of a ferret infected with NL/230 virus (B). No virus staining was present in the conjunctiva (C) or eye (D) of control ferrets.
Figure 4
Figure 4. Virus deposition in ferrets following different routes of inoculation.
Fluorescent-labeled A/NL/219/03 virus (NL219-FL) was administered to ferrets by the intranasal or ocular route. Each ferret was euthanized 15 min following virus administration and organs were collected for ex vivo imaging. Nasal turbinates are contained within the cap; left and right conjunctiva and eyes are below, respectively. An increasing fluorescence signal is indicated by brightness from red to yellow. Images are representative of triplicate independent inoculations for each route. Percentages represent the mean maximum relative efficiency for each tissue (n = 3) above levels in corresponding naïve tissue for each route of inoculation.
Figure 5
Figure 5. Transmissibility of influenza viruses in ferrets following ocular inoculation.
Three ferrets were inoculated by the ocular route with 106 EID50 of NY/107, NL/230, Brisbane or Panama virus, and nasal washes were collected from each ferret on the indicated day p.i. (solid bars). A naïve ferret was placed either in the same cage (A) or in an adjacent cage with perforated side walls (B) as each inoculated ferret 24 hrs p.i., and nasal washes were collected from each contact ferret on indicated days p.c. (hatched bars) to assess virus transmission in the presence of direct contact or respiratory droplets, respectively. The limit of virus detection was 101.5 EID50/ml.
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