*4.3. Inactivation of CSFV by Treatment with Tween20 and Heat*

The first inactivation experiment was performed using infectious cell culture supernatants with high viral loads (TCID50 > 106/50 μL), two highly (CSF0947 and CSF0634) and two moderately virulent strains (CSF0902 and CSF0104), independently. Infectious cell culture supernatants were 1:2 diluted in PBS containing 0.05% Tween20, which represents a commonly used Tween20 concentration in blocking reagents and washing buffers of antibody ELISA kits. The diluted samples were incubated at 56 ◦C for 30 min in a water bath. Immediately after heat treatment, virus infectivity was quantified by sample titration on CSFV-susceptible cells according to the EU Diagnostic Manual for CSF Diagnosis, Technical Part [16], in three independent runs, and virus isolation as well as qRT-PCR were performed. Two PBS controls were included—for the first, the samples were 1:2 diluted in PBS and incubated at 56 ◦C for 30 min (PBS + H control), whereas the second control was not treated with heat (PBS control).

Additionally, CSFV inactivation in serum samples was investigated. Four serum samples containing infectious CSFV (2.14 <sup>×</sup> <sup>10</sup><sup>2</sup> to 3.77 <sup>×</sup> <sup>10</sup><sup>4</sup> TCID50/ <sup>50</sup> <sup>μ</sup>L) were 1:2 diluted in PBS supplemented with different Tween20 concentrations (0.05%, 0.1%, 0.3%, 0.5%, 1%, and 2%) and incubated at 56 ◦C for 30 min in a water bath. As described above, two PBS controls were included and the samples and were analyzed by virus titration, virus isolation, and qRT-PCR, as described above.
