Emem media

The human neuroblastoma-derived cell line SHSY5Y (ATCC CRL-2266; ATCC, Gaithersburg, MD, USA), a popular albeit imperfect neuronal model, was cultured and expanded for experiments in 42% Ham’s F12 media (Sigma Aldrich), plus 42% EMEM media (Sigma Aldrich), 15% fetal bovine serum, 1% penicillin-streptomycin, and 1% non-essential amino acids, with enzymatic passaging using TrypLE Select (Thermo Fisher Scientific). SHSY5Y neural cells were plated onto 96-well culture plates at 25,000 cells/well and grown for 1–2 days before treatments were applied.

A European strain of KHV, K250, isolated from a KHV outbreak in the UK in 2007, was propagated in the common carp brain (CCB) derived cell line (ECACC 10072802) at 20 °C in EMEM media (Sigma, Gillingham, UK) supplemented with 2 mM Glutamine, 1% non-essential amino acids (Sigma, Gillingham, UK), 2% Foetal Bovine Serum (FBS), and 10 mM HEPES (Sigma, Gillingham, UK) [56 ].
The supernatant of cells showing cytopathic effects was harvested, clarified by centrifugation at 4000× g for 15 min, and stored at 4 °C.
To determine the viral dose, the copy number of the KHV orf90 gene was quantified in a Taqman qPCR test previously described [8 (link)]. Viral nucleocapsid was extracted from the clarified supernatant using the EZ1 Virus Mini Kit and the EZ1 extraction robot (Qiagen, Manchester, UK) following the manufacturer’s instructions. To generate a standard curve, a fragment of 1450 bp of the KHV orf90 gene containing the qPCR region was cloned into the pGem-T Easy plasmid vector (Promega, Southampton, UK) as described before [57 (link)]. The template (dsDNA) copy number was calculated using a QuantiFluor dsDNA kit in a Quantus fluorimeter (Promega, Southampton, UK), and a plasmid dilution series, from 10⁶ to 1 copy, was generated.