Table 1.
Replication and respiratory droplet transmission of PN99 and IN11 influenza viruses in ferrets housed at different environmental conditions.
Fig 1.
Morbidity observed in influenza virus infected and uninfected ferrets.
Six ferrets each were housed under the designated environmental conditions for the purpose of respiratory droplet transmission experimentation. Three ferrets each were presented with 103.8–105.5 pfu of PN99 or IN11 virus by aerosol inhalation and contact ferrets, n = 3, were placed in adjacent cages one day later. Inoculated animals, n = 3, were monitored for changes in minute volume by plethysmography (A) and weight (B) on the days noted. Endpoint (EP) data were collected on days 19–29 post inoculation. Both infected (C) and uninfected (D) contact animals were monitored for weight loss. Any contact animal shedding detectable virus in nasal washes and sero-converting to homologous virus was considered infected. EP data were collected on days 18–28 post contact. Mean values ± SEM is shown.
Fig 2.
Respiratory droplet transmission of PN99 and IN11 influenza virus among ferrets.
Three ferrets each were housed under the designated environmental conditions and were presented with 103.8–105.5 pfu of PN99 (A) or IN11 (B) virus by aerosol inhalation. One day later, a naïve, contact ferret was placed in a cage adjacent to each inoculated animal. Nasal washes were collected from ferrets every two days for up to 11 days and virus titers were assessed by plaque assay. Solid bars represent data from inoculated ferrets, n = 3, and hatched bars represent contact ferret data. The mean ± SD is shown and the limit of virus detection is 100pfu/mL. Frequency of transmission events is shown as an inset on each panel and represents the number of contact animals that shed virus and seroconverted.
Fig 3.
Size distribution of aerosols exhaled from influenza virus infected ferrets.
Aerosol particle counts were measured for individual ferrets, n = 3, collected during 15 minutes of normal breathing (A) or 5 minutes of sneezing stimulation (B) on 2, 4 and 6 dpi. Ferrets were housed under controlled environmental conditions as indicated. Aerosol particles within the range of 0.5μm to 20 μm in size are shown with key sizes noted on the x axis and a broken vertical line denoting the respirable particle fraction at <5 μm.
Fig 4.
Volume of aerosols exhaled by naïve and influenza virus inoculated ferrets.
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.
Fig 5.
Influenza virus detection in aerosol samples exhaled by infected ferrets.
Three ferrets each were housed under the designated environmental conditions and were presented with 103.8–105.5 pfu of PN99 (green) or IN11 (orange) virus by aerosol inhalation. On 1, 3 and 5 dpi, aerosol samples were collected from ferrets for 15 minutes of normal breathing (A) and 5 minutes of sneezing stimulation (B) and were segregated based on size (0.65–4.7 μm or >4.7 μm) and then assayed for the presence of infectious influenza virus. Total plaque forming units (pfu) from individual ferrets, n = 3, is shown with the grand mean for each sampling condition.
Fig 6.
Influenza virus RNA detection in aerosol samples exhaled by infected ferrets.
Three ferrets each were housed under the designated environmental conditions and were presented with 103.8–105.5 pfu of PN99 (green) or IN11 (orange) virus by aerosol inhalation. On 1, 3 and 5 dpi, aerosol samples were collected from ferrets for 15 minutes of normal breathing (A) and 5 minutes of sneezing stimulation (B) and were segregated based on size (0.65–4.7 μm or >4.7 μm) and then assayed for the presence of influenza virus RNA copies. RNA copy data from the three time points are combined and presented +SEM for each group.
Fig 7.
Recovery rates of influenza virus subjected to the aerosol collection procedure.
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.
Fig 8.
Influenza virus in aerosol samples exhaled by infected ferrets based on recovery rates.
Three ferrets each were housed under the designated environmental conditions and were presented with 103.8–105.5 pfu of PN99 (green) or IN11 (orange) virus by aerosol inhalation. Aerosol samples were collected from ferrets on 1, 3, and 5 dpi for 15 minutes of normal breathing (A) and 5 minutes of sneezing stimulation (B). Total plaque forming units (pfu) collected from infected ferrets were normalized based on the recovery rates of known amounts of infectious virus using our aerosol collection procedure. Total pfu exhaled by infected ferrets in both size ranges combined and at each time point are presented. Each dot represents a single animal at a single time point. Scatter dot plots show the distribution of data with the horizontal line representing the grand mean for all samples collected under the designated condition.