Comparative viral pathogenesis between intranasal and ocular inoculation day 3 p.i. in extra-ocular tissue.

a<p>All viral titers expressed per gram of tissue except NW, NT, and RS samples which are expressed per ml. Limit of detection is 1.5 log₁₀ EID₅₀. The mean viral titer of all ferrets with positive virus isolation (denoted in parentheses) is shown.</p>b<p>Route of inoculation. i.n., intranasal (10⁶ EID₅₀/ml) unless otherwise specified; i.o.; ocular inoculation (10⁶ EID₅₀/100 µl).</p>c<p>i.o. NW and RS samples are inclusive of 6 ferrets tested.</p>d<p>Viral titers represent a pooled intestinal sample consisting of the duodenum, jejuno-ileal loop, and descending colon.</p>e<p>Intranasal inoculation performed using 10⁶ EID₅₀/100 µl virus dose.</p>f<p>ND, not detected.</p

Virus deposition in ferrets following different routes of inoculation.

<p>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 <i>ex vivo</i> 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.</p

Comparison of mean titers of influenza viruses recovered from nasal wash following ocular inoculation of ferrets.

<p>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 10^1.5 EID₅₀/ml. †, ferrets did not survive to day 9 p.i.</p

Comparison of influenza virus recovery in conjunctival wash samples following ocular inoculation of ferrets.

<p>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 10^1.5 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.</p

Incidence of viral replication in ferrets following 10⁶ EID₅₀ ocular inoculation.

a<p>Limit of virus detection in nasal wash (NW) and rectal swab (RS) was 10^1.5 EID₅₀/ml, conjunctival wash (CW) was 10^0.8 EID₅₀/ml.</p>b<p>Titer of ferrets with positive virus isolation expressed as log₁₀ EID₅₀/ml ± standard deviation.</p>c<p>ND, not detected.</p

Transmissibility of influenza viruses in ferrets following ocular inoculation.

<p>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 10^1.5 EID₅₀/ml.</p

Volume of aerosols exhaled by naïve and influenza virus inoculated ferrets.

<p>Aerosol volumes were measured from ferrets during 15 minutes of normal breathing (NB) or 5 minutes of sneezing stimulation (SZ). Data collected from naïve animals, n = 6 (A), and data collected from inoculated animals (normalized to each ferret’s naïve level of aerosol shedding), n = 3, on 2, 4 and 6 dpi were combined and compared between PN99 and IN11 virus groups for aerosols <5 μm and ≥5 μm (B,C). Ferrets were housed under controlled environmental conditions as indicated. Data are presented + standard deviation.</p

Recovery rates of influenza virus subjected to the aerosol collection procedure.

<p>Prepared impactor plates were spiked with 10²–10³ pfu of PN99 (green) or IN11 (orange) influenza virus and were placed at a designated environmental condition as shown on the x-axis while air was pulled through them for 15 minutes (A) or 5 minutes (B). Experiments were performed in duplicate and the percentage of recovered RNA was determined by real time RT-PCR using M gene primers and infectious virus recovery was based on plaque assays. Each shaded section represents the proportion of the total amount of input virus that was recovered.</p

Summary of virus pathotyping in ferrets following 10⁶ EID₅₀ ocular inoculation.

a<p>Pathogenicity phenotype using the Intravenous Pathogenicity Index (IVPI) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002569#ppat.1002569-OIE1" target="_blank">[60]</a>. HPAI, highly pathogenic avian influenza; LPAI, low pathogenic avian influenza.</p>b<p>As determined by isolation of virus from samples collected during observation and seroconversion at the end of the experiment.</p>c<p>Average of peak mean change among ferrets from which virus was isolated from samples collected during observation.</p

Comparative viral pathogenesis between intranasal and ocular inoculation day 3 p.i. in ocular tissue.

a<p>Limit of detection is 1.5 log₁₀ EID₅₀ (eye and conj) or 0.8 log₁₀ EID₅₀ (CW, all 100 µl i.n. samples). The mean viral titer of all ferrets with positive virus isolation (denoted in parentheses) is shown.</p>b<p>Route of inoculation. i.n., intranasal (10⁶ EID₅₀/ml) unless otherwise specified; i.o.; ocular inoculation (10⁶ EID₅₀/100 µl).</p>c<p>Relative viral RNA copy number in CW samples determined by RT-PCR and extrapolated using a standard curve of known virus. i.o. CW samples are inclusive of 6 ferrets tested.</p>d<p>Intranasal inoculation performed using 10⁶ EID₅₀/100 µl virus dose.</p>e<p>ND, not detected.</p