*4.4. Validation of the Inactivation Protocol*

The protocol for inactivation was validated using a panel consisting of 33 serum samples with different virological and serological properties. To confirm virus inactivation, the samples were treated with PBS 0.3% Tween20 and heat (56 ◦C for 30 min), and thereafter subjected to virus isolation. Subsequently, serological analysis (VNT, CSFV Ab ELISA (IDEXX) and *pigtype*® CSF Erns AB ELISA (Indicial)) were performed. To consider a safety margin regarding virus inactivation, the samples were analyzed after treatment with PBS 0.5% Tween20 and also heat. For comparison, untreated serum samples were also analyzed.

#### *4.5. Virus Isolation*

Virus isolation was performed using the following three sample set-ups: (1) four infectious cell culture supernatants treated with heat and PBS 0.05% Tween20 or PBS (PBS control), (2) four serum samples treated with heat and PBS 0.3% Tween20 or PBS (PBS control), and (3) serum samples of category I out of the serum validation panel that were diluted in PBS 0.3% Tween20 and incubated for 30 min at 56 ◦C. The protocol for virus isolation followed the instructions described in the EU Diagnostic Manual for CSF Diagnosis, Technical Part [16]. Briefly, two concentrations of the serum samples (undiluted and 1:10 diluted) were incubated on PK15 and SK6 cells for three days, respectively. A positive and negative control was treated equally to the sample material. After two passages, cell culture supernatant was removed and the cells were fixed by heat treatment (80 ◦C for 3 h). Subsequently, an indirect immune-peroxidase staining was performed using the NS3-specific monoclonal mouse antibody BVD/C16 (dilution 1:25 in PBS-0.01%Tween20) and a polyclonal rabbit anti-mouse horseradish peroxidase conjugate (dilution 1:200 in PBS-0.01% Tween20, DAKO, Denmark).
