TDK conference 2020

The porcine reproductive and respiratory syndrome (PRRS) is one of the diseases that nowadays cause the greatest economic losses in pig farming. The disease is caused by Betaarterivirus suid species 1 and 2 (PRRSV 1, 2) belonging to the family Arteriviridae of the order Nidovirales.

The clinical symptoms caused by PRRSV are various in different age groups. In the case of sows, reproductive disorders occur, and when newborn piglets are infected, vitality decreases and respiratory symptoms appear, which significantly increases neonatal mortality. Severe respiratory symptoms and pneumonia occur in the adolescent and obese age groups. The virus has immunosupressive properties by targeting mainly the alveolar macrophages of the lung and similar cells of the lymphoid organs. The course of the disease is highly dependent on the virulence of the particular virus strain, as more severe lesions, different TNFα (tumor necrosis factor-α) and interleukin-10 expression have been observed during infection with strains with higher pathogenicity compared to strains with lower pathogenicity. The main goal of the present study was to develop a multiplex fluorescent in situ hybridization method that can be used to simultaneously examine the tissue expression of inflammatory cytokines and PRRSV in one section. Hence, piglets were infected with two PRRSV strains possessing different pathogenicity and slaughtered at different time points after infection. Thymic, tracheobronchial lymph node and lung samples were fixed in formalin, dehydrated, embedded in paraffin, and then 3 µm sections were prepared. Following deparaffinization and cell digestion, TNFα and IL-10 cytokines were identified simultaneously in sections using a multiplex fluorescent in situ hybridization method (RNAscope) with complementary probes designed for their mRNAs, and the RNA encoding the PRRSV nucleocapside was also displayed by a third probe. Tissue sections were digitized using a section scanner (Pannoramic MIDI II, 3D Histech) and then examined at 4 different wavelengths corresponding to the fluorophore dyes used. In case of using fluorescent labeling systems, especially for paraffin-embedded samples, the autofluorescence of the sample can often deliver some problems. To solve this issue, various autofluorescence suppression methods (longer deparaffinization, use of quencher solution, photobleaching) were also investigated to successfully optimize the RNAscope method at the applied wavelengths. Using the established assay, we can better understand the pathogenesis of PRRS disease and the nature of immunobiological processes caused by different strains of the virus. The successfully optimized multiplex fluorescent in situ hybridization process can serve as a useful novel tool in veterinary science.