Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo)

Figure 1. Clinical features of ferrets infected with EV-D68 virus. 12 EV-D68-infected ferrets (104.5CCID50 per animal) and 3 mock-infected ferrets (equivalent volumes of virus-free DMEM) were monitored daily for clinical features. The data were recorded as percentage changes compared with value of zero days post-infection. (A) The body temperature changes of infected and uninfected animals. Data is mean of rectal temperatures of each ferret groups of 3 to 12 ferrets per time point. The error bars show the SEM of temperature changes at different time; (B) The body weight changes of infected and uninfected animals. Data is mean of each groups of 3 to 12 ferrets per time point. The error bars show the SEM of temperature changes at different time; (C) The complete blood count (CBC) analysis of mock and EV-D68-infected ferrets. The percentages of lymphocytes, monocytes, neutrophils, and eosinophils were plotted respectively. Blood samples were collected at three, five, seven, and nine days post-infection. All samples were run in triplicate, with mean value and SEM. Monocytes: *, p < 0.05, EV-D68-infectedferrets compared with mock on five days post-infection; five days post-infected ferrets…

Figure 2. Dynamic distribution of EV-D68 virus in infected ferrets through respiratory route. Twelve ferrets were infected with EV-D68 (104.5CCID50/ferret) via nostrils dropwise and three ferrets set as mock control. Viral load in feces (A) and nasal washes (B) and throat swabs (C) of infected ferrets were detected in the whole course of EV-D68 infection (1, 3, 5, 7, 9, 11, 13, 15 days post-infection); (D) Viral RNA was picked up from blood samples and analyzed by Taqman real-time quantitative PCR assay, according to the protocols provided by the qPCR kit. Each blood samples were gathered during the whole EV-D68 infection course (1, 3, 5, 7, 9, 11, 13, 15 days post-infection); (E)Viral load in lung of ferret at different day post-infection (3, 5, 7, 9 days post-infection); (F) Viral load in lymph nodes at different day post-infection(3, 5, 7, 9 days post-infection). The vitro-synthesized RNA was applied to quantify the viral copies of each RNA sample, and viral RNA’s relative copies for each sample was calculated by the mathematical formula as follow: [(μg of RNA/μL)/(molecular weight)] × Avogadro’s number = viral copy number/μL. A viral load which is less than 10 copies is regarded as…

Figure 3. Pathological manifestations in respiratory apparatus of EV-D68-infected ferrets. (A) The clinical pathological features of EV-D68 infection in ferret lung tissues; (B) There is no pathological change in trachea. In these images, the white arrow represents the inflammatory cell infiltration, the black arrow points the diffuse alveolar hemorrhage. The microscope magnified the images 200 times, Bar, 100 µm; (C) The viral antigen expression in lung tissue from ferrets infected by 104.5CCID50 EV-D68. The black dashed boxes indicate the EV-D68 VP1 antigen expression and the insert picture enlarge the antigen (on the top right corner); (D) There is no VP1 antigen detectable in trachea. Images are magnified by 400 times; Bar, 50 μm.

Figure 4. Confocal imaging of α2,6-linked SAs and viral antigen in the lung tissue of EV-D68-infected ferrets. The samples were obtained at three and seven days post-infection. EV-D68 VP1 antigen was labeled with Texas-Red-conjugated anti-IgG antibody (red), while α2,6-linked SAs were labeled with FITC (green). Images are shown at 630× magnification.

Figure 5. Inflammatory cytokines detection of the ferrets after EV-D68 infection. (A) Cytokines (IL-1a, IL-5, IL-8) of serum were determined using multiplex bead-based Bio-Plex assay and are detected at different day (0, 5, 7, 14) post-infection; (B) The average transcription levels of interleukin-1α (IL-1α), IL-5, IL-8 of lungs were determined by qPCR and are plotted graphically for various time points (0, 5, 7, 9) following infection. Increases of mRNA levels were relative to β-actin and then normalized to the PBS control groups; (C) Cytokines (IL-12, IL-13, IL-17a) of serum were determined using multiplex bead-based Bio-Plex assay and are detected at different days (0, 5, 7, 14) post-infection; (D) The average transcription levels of IL-12, IL-13, IL-17a of lungs were determined by qPCR and are plotted graphically for various time points (0, 5, 7, 9) following infection. Increases of mRNA levels were relative to β-actin and then normalized to the PBS control groups. Average data were obtained from three independent experiments, and the error bars indicate SEM. The dot line represents the mRNA base level. Horizontal bars show the statistical analysis performed between the selected two…

Figure 6. The neutralization antibody titer of ferrets against the EV-D68 virus. Each plot represents the average of three logarithmically transformed measurements (log2).Vertical bars represent the mean value ± SEM. The number under each figure represents the timing of blood collection at different days after the inoculation.