Table 1.
Scoring criteria for lung function by auscultation.
Table 2.
Histologic lung lesion scoring criteria.
Fig 1.
Respiratory distress score of lambs inoculated with human respiratory syncytial virus (hRSV) strain Memphis 37 (M37).
Respiratory distress score was assessed daily for each lamb by auscultation or visual observation, and was categorized by expiratory effort (A) and wheezing (B). Results are shown as mean ± standard error.
Fig 2.
Viral gross lesions caused by M37 hRSV infection in neonatal lambs.
(A) Picture of a lung on day 6 post-infection. Dark plum-red, well-demarcated foci of pulmonary consolidation are indicated by arrowheads. (B) Percentage parenchymal involvement was estimated for each lung lobe and mean percentage averages per lobe were calculated for each day of necropsy (± standard error). Legend: Rt Cr = Right cranial lobe; Rt Mid = Right Middle lobe; Rt Cd = Right Caudal lobe; Acc = Accessory lobe; Lt Cr = Left Cranial lobe; Lt Mid = Left Middle lobe; Lt Cd = Left Caudal lobe.
Fig 3.
Microscopic lung lesions severity score in M37 hRSV infected neonatal lambs.
(A) Histopathologic lesions included bronchiolitis with degenerate/necrotic individual epithelial cells (thin arrow), occasional syncytial cells (long arrow), accumulation of degenerate neutrophils (short arrow), and occasional macrophages. H&E Bar = 50 μm. (B) A histologic score was given by determining percent consolidation followed by conversion to an integer-based consolidation scale used by our laboratory previously [1]: 0% consolidation = 0; 1%-9% consolidation = 1; 10%-39% consolidation = 2; 40%-69% consolidation = 3; 70%-100% consolidation = 4. Group averages were calculated for alveolar and bronchiolar consolidation scores. In addition to the consolidation score, bronchitis, bronchiolitis, neutrophil infiltration, peribronchiolar and perivascular infiltration of lymphocytes, syncytial cell formation, and epithelial alterations were also scored. Results are indicated as mean ± standard error for each scored parameter.
Table 3.
Quantification of RSV replication via RT-qPCR and infectious focus assay in lambs inoculated with M37 hRSV.
Fig 4.
Immunohistochemistry and scoring of RSV antigen expression in lambs inoculated with M37 hRSV.
Immunohistochemistry was used to detect viral antigen using an all-antigens polyclonal antibody for RSV. (A) RSV immunoreactivity is shown within epithelial cells lining the bronchioles (brown cells). Bar = 50 μm (B) The mean number of virally-infected bronchi/bronchioles and alveoli per field was counted for each day of necropsy.
Fig 5.
Lung chemokine mRNA expression assessed by RT-qPCR in lambs inoculated with M37 hRSV.
Lung tissue obtained from each animal was evaluated for the following mRNA targets: surfactant protein A (SP-A), surfactant protein D (SP-D), interleukin 8 (IL-8), interleukin 10 (IL-10), macrophage inflammatory protein 1 alpha (MIP-1α), monocyte chemotactic protein 1 alpha (MCP-1α), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interferon beta (IFN-β), interferon gamma (IFN-γ), programmed cell death 1 ligand 1 (PD-L1), and regulated on activation normal T-cell expressed and secreted (RANTES). Mean relative mRNA expression was calculated for each target with respect to each day of necropsy. Relative mRNA expression means: relative to the total amount of RNA loaded per reaction (which is kept constant) and relative to the values established by the standard curves for each target.