In Situ Hybridization Detection of Bovine Respiratory Syncytial Virus in the Lung of Experimentally Infected Lambs

Abstract

We studied the distribution of bovine respiratory syncytial virus (BRSV) RNA in lungs of experimentally inoculated lambs by in situ hybridization at different times postinoculation. The probe used for in situ hybridization was prepared by reverse transcription of BRSV RNA, followed by polymerase chain reaction (PCR) amplification of the cDNA. Twenty-five Merino lambs of both sexes with a live weight of 17 +/- 3 kg received an intratracheal inoculation of 20 ml saline solution containing 1.26 X 10(6) TCID50 BRSV (strain NMK7)/ml. Lambs were slaughtered 1, 3, 7, 11, and 15 days postinoculation (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 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. Mononuclear cells were located in peribronchiolar tissue and interalveolar septa. The highest signal intensity in positive cells was observed at 3 and 7 PID, coinciding with the most important histopathological findings.

Investigation of respiratory syncytial virus (RSV) and parainfluenza-3 (PI-3) virus by histopathological and immunohistochemical methods in sheep and goat

“…In previous studies, it was determined that the areas where BRSV was detected in the lung by the immunohistochemical method were virus replication areas (Masot et al, 2000). In studies conducted with natural and experimental RSV infection of ruminants (Ceribasi et al, 2014;Jarikre and Emikpe, 2017;Masot et al, 2000;Viuff et al, 1996;Yener et al, 2005), bronchiolar epithelial cells were determined by the IHC method in type II pneumocytes, epithelial cells of bronchial glands, syncytial cells, alveolar macrophages, and exudate in the bronchial, bronchiolar, and alveoli lumen. In the study, it was determined that the intensity of RSV antigen staining in sheep and goats was statistically similar in bronchial and bronchiolar epithelial cells and cell debris, peribronchial glands and interalveolar inflammatory cells infiltrations.…”

Epidemiology, Molecular Epidemiology and Evolution of Bovine Respiratory Syncytial Virus

“…Regardless of the geographical location, infectivity rates are usually rather high, suggesting that viral transmission is a common event among herds. Cattle are the principal reservoir of infection; however, sheep can also become infected [7]. Intra-herd transmission usually occurs by aerosols, allowing the virus to enter susceptible cattle via the respiratory tract.…”

Respiratory syncytial virus nephropathy in rats

“…In situ hybridization RSV-specific probes composed of three oligonucleotides with sequences chosen from the published sequence of the G protein mRNA of RSV (sequence I: 5 0 -cttttctagtgtcttagcggtgcgttggtccttg-3 0 ; sequence II: 5 0 -gcaggtttgacttgactcctggtgttgttgaagc-3 0 ; sequence III: 5 0 -ctcttctgtgggcttggtggtgggtacttccttt-3 0 ) were synthesized by Genebase Biology Company (Shanghai, China). The oligonucleotides were end-labelled with digoxigenin and hybridization was carried out by a hybridization detection kit according to the manufacturer's instruction (DakoCytomation, Denmark) and reference on in situ hybridization, 31 with minor modifications. Kidney and lung sections (5 mm thick) were deparaffinized with xylene, rehydrated through decreasing concentrations (100-95-85-70%) of ethanol, inactivated with endogenous enzymes by 3% hydrogen peroxide at room temperature for 5-10 min, and digested with 0.1 mg/ml proteinase K at 371C for 10 min.…”