IPN in Vitro Challenge
IPN in Vitro Challenge
To establish a primary hepatocyte culture, 200 g fish from the Fish Centre facility at NMBU were euthanized by a sharp blow to the head, and the hepatocytes were isolated as previously described: Datsomor, A.K.; Wilberg, R.; Torgersen, J.S.; Sandve, S.R.; Harvey, T.N. Efficient Transfection of Atlantic Salmon Primary Hepatocyte Cells for Functional Assays and Gene Editing. G3 Genes|Genomes|Genetics 2023 Liver cells were cultured at 20°C in hepatocyte culture media for a period of two days. After that time, the cells were washed with PBS and fresh culture media was added supplemented additionally with growth factors (5 ng/mL epidermal growth factor (EGF) and 3 ng/mL hepatocyte growth factor (HGF)). At day 7 post-culture, the media was replaced once again to hepatocyte culture media without growth factors. Two independent challenge tests using primary hepatocytes from 4 different fish (F1 – F4) were performed. Challenge 1 consisted of cells from F1 while the hepatocytes from the remaining fish (F2 – F4) were part of challenge 2. The cells from both challenges were infected in duplicates at MOI 0.1 with cIPNV or rIPNV for 1 hour at 15°C. After incubation time, the viral inoculum was removed and replaced with infection media containing also Amphotericin B (2.5 µg/mL). The cells were kept at 15°C and monitored daily for CPE. Both cells and supernatants were collected for RNA extraction at 4 and 7 dpi for challenge 1 and at 2 and 4 dpi for challenge 2. total RNA was extracted using the RNeasy Mini kit (Qiagen®, following the manufacturer's protocol for extraction from cells. he RNA samples were labelled and stored at -80°C until processed downstream. Libraries were constructed by NovoGene and BmkGene