Bovine respiratory syncytial virus in-situ hybridization from sheep lungs at different times postinfection

We studied the distribution of bovine respiratory syncytial virus (BRSV) RNA in lungs of experimentally infected sheep by in situ-hybridization at different times postinfection. The probe used for in-situ hybridization was prepared by reverse transcription of BRSV RNA, followed by PCR amplification of the cDNA. Twenty five Merino sheeps of both sexes with a live weight of 55± 10 Kg, received a intratracheal inoculation of 40 ml saline solution containing 1.26 × 10⁶ TCID₅₀ BRSV (strain NMK7) per ml. Sheep were slaughtered 1,3,7,11 and 15 postinoculation days (PID). Bronchial and bronchiolar epithelial cells were positive for BRSV nucleic acid by ISH at 1, 3, 7 and 11 PID. However, alveolar epithelial cells contained positive hybridization signals cells at 1,3 and 7 PID. Cells containing viral RNA were detected from 1 to 11 PID, in exudate within bronchial and bronchiolar lumina; and from 3 to 7 PID in alveolar exudates. Positive hybridization signals were identified in interstitial mononuclear cells and in bronchi associated lymphoid tissue from 3 to 11 PID. The highest signal intensity of positive cells were observed at 3 and 7 PID, coinciding with high virus antibodies titres and with the most important histopathological findings.