Crl 2747

The ISAV-Glesvær isolate [21 ] was propagated in an ASK cell line (ATCC^® CRL-2747™) as previously described [22 (link)]. Briefly, cells were grown at 20 °C in T-150 culture flasks containing Leibowitz L-15 medium (Thermo Fisher Scientific, Warrington, United Kingdom) supplemented with 10% fetal bovine serum (FBS) and gentamicin (Lonza, USA). The ISAV isolate was used to inoculate the cell culture and incubated for seven days at 15 °C. Cell culture supernatant was harvested and clarified by centrifugation at 3800 g for 10 min at 4 °C. The clarified supernatant in 1 mL aliquots was frozen at −80 °C. Virus titer was calculated according to the 50% end-point method of Reed and Muench (1938) [23 (link)], and expressed as the dilution of the virus causing 50% tissue infection per milliliter (TCID₅₀/mL).

Atlantic salmon kidney cells (ASK) were acquired from the ATCC (ATCC® CRL-2747) (Devold et al., 2000 (link)). Monolayers of ASK were maintained at 15°C in Leibovitz medium (L-15, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), gentamycin (50 μg/ml, US Biological) and β-mercaptoethanol (55 mM, Bio-Rad, USA). A viral inoculum stock of ISAV genotype HPR35, with a titer of TCID50 = 10⁷/mL, was kindly provided by ADL diagnostics. The virus was propagated in monolayers of 70% confluent ASK cells as described in the viral infection section. All supernatants of the culture (aliquots of virus) were stored at −80°C.