*4.6. ELISAs for Detection of Antibodies Against CSFV*

For antibody detection against CSFV, two commercially available ELISAs were carried out according to the manufactures instructions. The antibody ELISAs are different with regard to antigen specificity, the CSFV Ab ELISA (IDEXX) detects antibodies against the structural glycoprotein E2 and the *pigtype*® CSFV Erns AB ELISA (Indicial) against the structural protein Erns.

#### *4.7. Virus Neutralization Assay*

The VNT has been performed according to the standard protocol described in the EU Diagnostic Manual for CSF Diagnosis, Technical Part [16], starting with a 1:5 dilution of the serum sample. The final applied dilution was 1:640. The cell line PK15 and the CSFV reference strain Diepholz (CSF0104; genotype 2.3) were used.

#### *4.8. Isolation of Viral RNA and qRT-PCR*

To analyze the influence of Tween20 on genome detection by qRT-PCR, total RNA was extracted from four serum samples and four infectious cell culture supernatants that were diluted in PBS 0.3% Tween20 along with incubation at 56 ◦C for 30 min or PBS without heat treatment (PBS control), independently. For this purpose, the QIAamp Viral RNA Mini Kit (Qiagen, Germany) was used according to the manufacturers' recommendations. Afterwards, viral RNA was detected by a CSFV-specific qRT-PCR, as described previously [17]. The samples were analyzed in duplicate.

**Author Contributions:** Conceptualization, D.M., A.P., and P.B.; methodology, D.M. and A.P.; validation, D.M. and A.P.; formal analysis, D.M., A.P., and P.B.; investigation, D.M. and A.P.; resources, P.B.; data curation, D.M. and A.P.; writing—original draft preparation, D.M.; writing—review and editing, D.M., A.P., and P.B.; visualization, D.M. and A.P.; supervision, P.B.; project administration, D.M., A.P., and P.B.; funding acquisition, P.B.

**Funding:** This research was funded by the Directorate General for Health and Food Safety of the European Commission, which provided the budget for the EU Reference Laboratory for Classical Swine Fever (Grant decision SI2.727610). This publication was supported by Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation within the funding programme Open Access Publishing.

**Acknowledgments:** We are grateful to Gabriele Müller, Karin Ruthenberg, and Inga Grotha for their excellent technical assistance. Furthermore, we would like to thank Sophia Austermann-Busch for fruitful discussions and support.

**Conflicts of Interest:** The authors declare no conflict of interest.
