Figure 5: Histopathology and cytokine/chemokine mRNA analyses in lungs from vaccinated cotton rats following RSV challenge.

(A) Lung tissues were collected at 5 days post-challenge with RSV-A2 and were stained with hematoxylin and eosin. Individual lungs were scored for pulmonary inflammation: peribronchiolitis (inflammatory cell infiltration around the bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). Slides were scored blindly using a 0–4 severity scale. The scores were subsequently converted to a 0–100% histopathology scales. (B–D) Cytokines and chemokines mRNA in lung homogenates from day 2 and day 5 post viral challenge were measured by RT-qPCR using primers specific for IFN-γ (B), IL-4 (C) or MCP-1 (D). The baseline cycles and cycle threshold (Ct) were calculated by the iQ5 software in the PCR Base Line Subtracted Curve Fit mode. The Ct values were plotted against Log10 cDNA dilution factor. These curves were used to convert the Ct values obtained for different samples to relative expression units. These relative expression units were then normalized to the level of β- actin mRNA (“housekeeping gene”) expressed in the corresponding sample. For animal studies, mRNA levels are expressed as the geometric mean ± SEM for all animals in a group at a given time.